Vasodilating agents and platelet function: intracellular free calcium concentration, cyclic nucleotides, and shape-change response.

J Cardiovasc Pharmacol (UNITED STATES) 1986, 8 Suppl 8 pS102-6

The adenylate-cyclase activator forskolin, the guanylate-cyclase stimulator sodium nitroprusside, the phosphodiesterase inhibitor Ro 15-2041, different Ca-entry blockers, as well as various vasodilators, and the atrial natriuretic peptide were tested for antiplatelet activity. Thrombin, vasopressin, ADP, arachidonic acid, and the dihydropyridine Ca agonist CGP 28392 were used as platelet activators. The physiological and biochemical parameters of platelet function studied included shape-change reaction, intracellular free-Ca modulation, and cyclic nucleotide formation. When inhibition of the shape-change response occurred, it was accompanied by inhibition of the increase in intracellular free Ca. Furthermore, the results suggest a possible intracellular site of action of Ca entry blockers in platelets, and confirm the importance of modulation of cyclic nucleotides in the regulation of platelet function, regardless of the mechanism of platelet activation. Additional antiplatelet activity of antihypertensive agents may have a beneficial effect in reducing the associated risk of thrombo-embolic complications in essential hypertension.

Comparison of calcium-current in isolated atrial myocytes from failing and nonfailing human hearts.

Mol Cell Biochem (NETHERLANDS) Apr 12-26 1996, 157 (1-2) p157-62

To identify possible alterations of the L-type calcium currents (I(Ca),L) in cardiomyopathy, I(Ca),L were recorded in atrial myocytes dissociated from the nonfailing heart (NF) of patients undergoing corrective open-heart surgery and explanted failing heart (FH) of patients with dilated cardiomyopathy undergoing heart transplantation. The patch-clamp technique was applied in the single-electrode whole-cell mode. The electrophysiological properties of I(Ca),L, including cell capacitance and current density, were similar in atrial myocytes from both groups of patients. Further to identify possible alterations of the myocardial beta-adrenergic pathway in cardiomyopathy, we examined the effects of isoproterenol,forskolin, 8-Br-cAMP and IBMX on I(Ca),L in both groups of atrial myocytes. Perfusion of isoproterenol (1 microM) significantly increased the peak I(Ca),L by 515 +/- 44% in 6 atrial myocytes from NF but increased only by 135 +/- 25% in 27 atrial myocytes from FH. However, forskolin (1 microM) or 8-Br-cAMP (0.1 mM) increased the peak I(Ca),L to a similar extent in atrial myocytes from NF and FH. IBMX (20 microM) also induced a comparable increase in the peak I(Ca),L by 213 +/- 31% (n = 5) and 207 +/- 59% (n = 4) in atrial myocytes from NF and FH, respectively. The above findings suggest that in atrial myocytes obtained from FH the beta-adrenoceptor numbers might be decreased but no impairment of the signal transduction cascade occurred beyond the GTP binding proteins level.

Differential changes in left and right ventricular adenylyl cyclase activities in congestive heart failure

American Journal of Physiology - Heart and Circulatory Physiology (USA), 1997, 272/2 41-2 (H884-H893)

The status of beta-adrenergic receptors and adenylyl cyclase in crude membranes from both left and right ventricles was examined when the left coronary artery in rats was occluded for 4, 8, and 16 wk. The adenylyl cyclase activity in the presence of isoproterenol was decreased in the uninfarcted (viable) left ventricle and increased in the right ventricle subsequent to myocardial infarction. The density of beta1-adrenergic receptors, unlike beta2-receptors, was reduced in the left ventricle, whereas no change in the characteristics of beta1- and beta2-adrenergic receptors was seen in the right ventricle. The catalytic activity of adenylyl cyclase was depressed in the viable left ventricle but was unchanged in the right ventricle. In comparison to sham controls, the basal, as well as NaF-, forskolin-, and 5'-guanylyl imidodiphosphate (Gpp(NH)p)-stimulated adenylyl cyclase activities were decreased in the left ventricle and increased in the right ventricle of the experimental animals. Opposite alterations in the adenylyl cyclase activities in left and right ventricles from infarcted animals were also seen when two types of purified sarcolemmal preparations were employed. These changes in adenylyl cyclase activities in the left and right ventricles were dependent on the degree of heart failure. Furthermore, adenosine 3',5'-cyclic monophosphate contents were higher in the right ventricle and lower in the left ventricle from infarcted animals injected with saline, isoproterenol, or forskolin in comparison to the controls. The results suggest differential changes in the viable left and right ventricles with respect to adenylyl cyclase activities during the development of congestive heart failure due to myocardial infarction.

Chronic opiate-receptor inhibition in experimental congestive heart failure in dogs

American Journal of Physiology - Heart and Circulatory Physiology (USA), 1997, 272/1 41-1 (H478-H484)

Acute administration of opiate-receptor antagonists has previously been shown to improve cardiac output, sortie blood pressure, systolic ventricular performance, and the baroreflex function in conscious dogs with right-sided congestive heart failure (RHF). However, whether similar changes occur after chronic opiate-receptor inhibition in congestive heart failure is not known. To determine the chronic effects of opiate-receptor antagonism on RHF, we administered naltrexone (200 mg/day), a long-acting, orally active opiate- receptor blocking agent, to RHF and sham-operated animals for 6 wk. Naltrexone had no effects on resting heart rate, right atrial pressure, aortic pressure, or cardiac output in RHF dogs but increased the first derivative of right and left ventricular pressure with respect to time (dP/dt) at rest and improved the dP/dt responses to isoproterenol. The inotropic responses to isoproterenol and forskolin in isolated right ventricular trabeculate muscle also were improved by chronic naltrexone in RHF. Myocardial beta-receptor density was reduced in the failing right ventricle compared with the control (58 plus or minus 3 vs. 108 plus or minus 6 fmol/mg protein, P < 0.01) but was unaffected by addition of naltrexone. Finally, naltrexone prevented the decline in baroreflex sensitivity that occurred in RHF (-0.2 plus or minus 0.5 vs. -6.0 plus or minus 0.5 ms/mmHg, P < 0.01). These effects of naltrexone did not occur in the sham-operated animals. Chronic opiate-receptor blockade with naltrexone attenuates the development of reduced adrenergic inotropic responsiveness and barereflex subsensitivity that occur in RHF. Because there was a similar improvement in the forskolin response in the absence of significant alterations in myocardial beta-adrenoceptor density after naltrexone treatment, the improvement in adrenergically mediated inotropic effects probably is mediated via a postreceptor mechanism.

beta-adrenoceptor mediated signal transduction in congestive heart failure in cardiomyopathic (UM-X7.1) hamsters

Molecular and Cellular Biochemistry (USA), 1996, 157/1-2 (191-196)

In view of the lack of information regarding the status of beta-adrenoceptor mediated signal transduction mechanisms at severe stages of congestive heart failure, the status of beta-adrenoceptors, G-proteins and adenylyl cyclase activities was examined in 220-275 day old cardiomyopathic hamster hearts. Although no changes in the Kd values for beta1- and beta2,-adrenoceptors were seen, the number of beta1-adrenoceptors, unlike that of beta2-adrenoceptors, was markedly decreased in cardiac membranes from failing hearts. The activation of adenylyl cyclase in the failing hearts by different concentrations of isoproterenol was also attenuated in comparison to the control preparations. The basal adenylyl cyclase activity in cardiac membranes from the failing hearts was not altered; however, the stimulated enzyme activities, when measured in the presence of forskolin, NaF or Gpp(NH)p were depressed significantly. The functional activity of Gs-proteins (measured by cholera toxin stimulation of adenylyl cyclase) was depressed whereas that of Gi-proteins (measured by pertussis toxin stimulation of adenylyl cyclase) was increased in the failing hearts. Not only were the Gs- and Gi-protein contents (measured by immunoblotting) increased, the bioactivities of these proteins as determined by ADP-ribosylations in the presence of cholera toxin and pertussis toxin, respectively, were also higher in failing hearts in comparison to the control values. Northern blot analysis revealed that the signals for Gs- and Gi-protein mRNAs were augmented at this stage of heart failure. These results indicate that the loss of adrenergic support at severe stages of congestive heart failure in cardiomyopathic hamsters may involve a reduction in the number of beta1-adrenoceptors, and an increase in Gi-protein contents as well as bioactivities in addition to an uncoupling of Gs-proteins from the catalytic site of adenylyl cyclase in cardiac membrane.

Pharmacology and inotropic potential of FORSKOLIN in the human heart.

J Clin Invest (UNITED STATES) Jul 1984, 74 (1) p212-23

We evaluated the effects of the diterpene compound FORSKOLIN in human myocardial adenylate cyclase preparations, isolated trabeculae and capillary muscles derived from failing human hearts, and acutely instrumented dogs. FORSKOLIN was a potent, powerful activator of human myocardial adenylate cyclase and produced maximal effects that were 4.82 (normally functioning left ventricle) and 6.13 (failing left ventricle) fold greater than isoproterenol. In contrast to isoproterenol, FORSKOLIN retained full activity in membrane preparations derived from failing hearts. In cyclase preparations, FORSKOLIN demonstrated unique substrate and Mg2+ kinetic properties that could be distinguished from hormone receptor-coupled agonists or fluoride ion. The adenylate cyclase stimulatory effect of FORSKOLIN was synergistic with isoproterenol, apparently due to the location of FORSKOLIN activation being beyond the level of hormone receptor-agonist in the receptor-cyclase complex. FORSKOLIN was a potent positive inotrope in failing human myocardium, producing a stimulation of contraction that was similar to isoproterenol. Finally, in open chest dogs FORSKOLIN was a positive inotropic agent that reduced preload and afterload. We conclude that FORSKOLIN belongs to a class of agents that may have therapeutic potential in the treatment of congestive heart failure.

[Effects of FORSKOLIN on canine congestive heart failure]

Nippon Yakurigaku Zasshi (JAPAN) Nov 1986, 88 (5) p389-94

FORSKOLIN is a diterpene of the labdane family which activates adenylate cyclase. The effects of FORSKOLIN were investigated in a congestive heart failure (CHF) model that we newly established using anesthetized dogs. The model was made by the intramural injection of protease into the left ventricular free wall, saline loading, and dextran and methoxamine infusion. By this maneuver, aortic blood flow (AoBF) was decreased; left atrial pressure (LAP), systemic vascular resistance (SVR) and left ventricular endodiastolic pressure (LVEDP) were markedly increased; and systemic blood pressure was unchanged. A bolus injection of 5.0 micrograms/kg FORSKOLIN reversed the hemodynamic findings of CHF. It reduced LAP (17.5----7.9 mmHg) (mean, N = 7), SVR (19980----10390 dyne sec/cm5), time constant T (90.7----59.2 msec) and LVEDP (22.8----16.8 mmHg); and it increased Vmax (2.32----2.82 l/sec) and AoBF (0.50----0.72 l/min). FORSKOLIN improved the CHF mainly through its vasodilator and positive inotropic actions.

Cultured human trabecular meshwork cells express functional alpha 2A adrenergic receptors.

Invest Ophthalmol Vis Sci (UNITED STATES) Nov 1996, 37 (12) p2426-33

PURPOSE: For the treatment of glaucoma, alpha-2 adrenergic receptor (alpha 2-AR) agonists are thought to lower intraocular pressure primarily by decreasing aqueous humor production. Effects on the outflow pathways, however, also may occur. To begin to examine this possibility, the authors characterized the alpha 2-AR subtypes present in cultures of human trabecular meshwork (HTM) cells using both immunofluorescence microscopy and functional measures of alpha 2-AR activation. METHODS: For immunofluorescence microscopy, subtype-specific polyclonal antibodies that recognize each of the human alpha 2-AR subtypes (alpha 2A, alpha 2B, alpha 2C) were used. Functional studies involved the inhibition of forskolin-stimulated cyclic adenosine monophosphate (cAMP) production, the stimulation of mitogen-activated protein (MAP) kinase activity, and the stimulation of mitotic activity as reflected by the expression of proliferating cell nuclear antigen (PCNA). RESULTS: From the immunofluorescence microscopy, there was evidence for the presence of the alpha 2A subtype, but not alpha 2B or alpha 2C subtype, on HTM cells. The administration of the alpha 2-agonist, dexmedetomidine, to HTM cells resulted in a 90% inhibition of forskolin-stimulated cAMP formation, a twofold stimulation of MAP kinase activity, and a threefold increase in the expression of PCNA. Additionally, preincubation of cells with either of the alpha 2-AR-selective antagonists, rauwolscine or atipamezole, reversed the functional effects of dexmedetomidine. CONCLUSIONS: Functional alpha 2A-ARs are present on HTM cells where they may affect the outflow pathway during the treatment of glaucoma with alpha 2-AR agonists.

Neurotransmitters and intraocular pressure

FUNDAM. CLIN. PHARMACOL. (France), 1988, 2/4 (305-325)

The role of the ocular autonomic nervous system in IOP regulation has been well established. Pharmacological and autohistoradiographic studies confirmed the high density of beta2 and alpha2 receptors on ciliary processes and iris epithelium. Their respective pharmacological activation or blockade is discussed. The role of other ocular neurotransmitters is also complex, as shown by the paradoxical similar action of dopamine agonists and antagonists on IOP. Concerning the cholinergic system, ocular muscarinic receptors are pharmacologically not well documented. Numerous other neurotransmitters may modulate IOP without necessarily leading to the development of new drugs. Drugs of the future will probably concentrate on dopaminergic agonists, cAMP-stimulators such as forskolin, prostaglandins, and cannabinoids.

HP 663: A novel compound for the treatment of glaucoma

DRUG DEV. RES. (USA), 1988, 12/3-4 (197-209)

Various studies suggest that the 7-dihydroxypropionyl derivative of forskolin, HP 663, effectively lowers intraocular pressure (IOP) by a novel mechanism. Biochemically, HP 663 stimulates rat striatal adenylate cyclase and displays an affinity for forskolin binding sites similar to that of forskolin. Following topical administration this compound lowers IOP in both New Zealand White (NZW) and Dutch Belt (DB) rabbits. In NZW rabbits, concentrations from 0.05 to 2.0% in a buffered hydroxypropylmethylcellulose (HPMC) vehicle produce significant reductions in outflow pressures of 35-45% for 5-6 hr. Additionally, HP 663 is soluble in this vehicle up to a concentration of 0.25%. As mentioned, HP 663 also reduces IOP in DB rabbits; however, this response is not significant until a concentration of 1% is attained. The effect on aqueous humor inflow was indirectly determined by studying the rate of IOP recovery following rapid i.v. infusion of 20% NaCl. In NZW rabbits, HP 663 significantly reduced aqueous humor inflow as shown by a 40% decrease in the IOP recovery rate compared to control. Tolerance also does not appear to develop to the IOP lowering effects of HP 663 in NZW rabbits. This compound is still capable of significantly lowering IOP following twice daily treatment for 21 consecutive days. Following topical administration of concentrations which effectively lower IOP, HP 663 exhibits no significant effects on heart rate or blood pressure of NZW rabbits. Therefore, this compound does not appear to exhibit a potential for peripheral side effects after topical application. Based on the results of the present study, HP 663 appears to be a potent activator of adenylate cyclase and may provide a novel and effective treatment for glaucoma

Intraocular pressure effects of multiple doses of drugs applied to glaucomatous monkey eyes

ARCH. OPHTHALMOL. (USA), 1987, 105/2 (249-252)

The effects of multiple dosing with 0.5% timolol maleate, 2% epinephrine hydrochloride, 4% pilocarpine hydrochloride, 1% vanadate, 1% forskolin (nonproprietary name, colforsin), or 0.5% prostaglandin F(2alpha) on intraocular pressure (IOP) were each tested on eight cynomolgus monkey eyes in which glaucoma was induced by photocoagulating the trabecular meshwork with the argon laser. The week prior to drug therapy, baseline IOP measurements were carried out at hourly intervals from 9:30 AM to 3:30 PM on three days. One to two days later, therapy was initiated. Each drug was applied topically to both eyes of each monkey twice daily for at least four days. The IOP was measured with a calibrated pneumatonometer at the same hourly intervals on treatment days as on the baseline days. The IOP at each time of day on treatment days was compared with the average baseline IOP measured at the corresponding time of day. Topical application of timolol, epinephrine, pilocarpine, vanadate, and prostaglandin F(2alpha) significantly reduced IOP without evidence of tolerance or tachyphylaxis during the course of therapy. Forskolin did not significantly decrease IOP after the second day of treatment.

Laser-induced glaucoma in rabbits

EXP. EYE RES. (UK), 1986, 43/6 (885-894)

Argon laser energy was applied to the trabecular meshwork of pigmented rabbits in an attempt to develop an animal model of 'glaucoma'. Laser energy was varied to determine the optimal level needed to produce sustained ocular hypertension. An initial response of ocular hypertension followed by hypotension was observed in all of the animals tested. Approximately half of the laser-treated rabbits developed a secondary buphthalmus and sustained ocular hypertension. In these animals outflow facility was decreased by approximately 60%. Histologic examination at 4-and 8 weeks after laser treatment demonstrated a wound-healing response resulting in closure of the intertrabecular spaces and obstruction of outflow to injected carbon particles. Optic nerve cupping and a loss of ganglion cells were also observed. Topical application of L-timolol (0.5%), pilocarpine (2.0%) and forskolin (1.0%) were found to be effective in decreasing intraocular pressure in the laser-treated, hypertensive eye with no significant effect in control non-laser-treated eyes, suggesting that this model can be a useful tool for screening potential antiglaucoma medications.

Regulation of aqueous flow by the adenylate cyclase receptor complex in the ciliary epithelium

AM. J. OPHTHALMOL. (USA), 1985, 100/1 (194-198)

The answer to how beta-adrenergic receptor mediates a fall in intraocular pressure has been elusive. Methods of measurement have not been refined sufficiently. The separate changes after adrenergic treatment frequently are small, and the tissue effects are multiple. On a molecular basis, stimulation of the beta-adrenergic receptor activates intracellular adenylate cyclase to produce increased cyclic adenosine monophosphate. Acting by different cell-receptor mechanisms, but nonetheless potent, nonadrenergic stimulators of adenylate cyclase in the ciliary epithelium, such as cholera toxin and organic fluorides, have been studied in experimental animals. They reduce intraocular pressure by reducing net aqueous flow. When forskolin, a diterpene and potent stimulator of adenylate cyclase, became available, it was used in noninvasive topical form in the human eye to clarify the question of whether increased cyclic adenosine monosphosphate reduces intraocular pressure and aqueous flow. Noninvasive studies in human eyes have demonstrated a 35% reduction in outflow pressure after the administration of forskolin in a 1% topical suspension, matched by a corresponding reduction in aqueous flow. Tonographic outflow facility was unaltered. Thus, the entire reduction in intraocular pressure can be accounted for by a reduction in net aqueous flow.

Forskolin lowers intraocular pressure by reducing aqueous inflow

INVEST. OPHTHALMOL. VISUAL SCI. (USA), 1984, 25/3 (268-276)

Forskolin is a diterpene derivative of the plant Coleus forskohlii that stimulates adenylate cyclase activity without interacting with cell surface receptors. Forskolin lowers the intraocular pressure of rabbits, monkeys, and humans. In rabbits, net aqueous humor inflow decreases, outflow facility remains unchanged, and ciliary blood flow increases. Tolerance to the intraocular pressure lowering effect did not occur in rabbits after topical doses given every 6 hr for 15 days. In vitro forskolin activates adenylate cyclase of crude particulate homogenates prepared from cultured human ciliary epithelia or from dissected ciliary epithelial processes of rabbit or human eyes. This activation is not blocked by timolol. The stimulation of adenylate cyclase by isoproterenol in vitro is potentiated in the presence of forskolin. Forskolin represents a potentially useful class of antiglaucoma agents differing in molecular mechanism of action from previously used drugs.

Indomethacin and epinephrine effects on outflow facility and cyclic adenosine monophosphate formation in monkeys

Investigative Ophthalmology and Visual Science (USA), 1996, 37/7 (1348-1359)

Purpose. To investigate the effect of indomethacin inhibition of prostanoid production on the epinephrine-stimulated increase in outflow facility and cyclic adenosine monophosphate (cAMP) production in the anterior segment of the monkey eye. Methods. Topical indomethacin was given 1 hour before the intracameral administration of epinephrine to living cynomolgus monkeys. Outflow facility was measured for 45 to 60 minutes, beginning 3 hours after epinephrine administration, by two-level constant pressure perfusion of the anterior chamber. Cyclic adenosine monophosphate formation was measured in cell membranes isolated from rhesus monkey ciliary muscle, ciliary muscle, ciliary processes, trabecular meshwork, and iris in the presence of forskolin, indomethacin, epinephrine, or indomethacin and epinephrine combined. Results. Three hours after the intracameral administration of 5.5 microg epinephrine, facility increased by similar40%, a putatively maximal response, at which time the intracameral epinephrine concentration was similar15 microM. Pretreatment with topical indomethacin produced a dose-dependent inhibition of epinephrine's facility-increasing effect; the maximum inhibition of 50% to 70% occurred at an indomethacin dose of 50 to 125 microg. Doubling the indomethacin dose (250 microg) produced no further inhibition, whereas a fivefold larger epinephrine dose (27.5 microg) did not overcome the inhibition. Forskolin and epinephrine both stimulated cAMP production in vitro, whereas (indomethacin) greater than or equal to 10-4 M partially inhibited both basal and epinephrine-stimulated cAMP production in all four tissues. Conclusions. Approximately half of the epinephrine-induced facility increase is inhibited by indomethacin, but it is unclear whether the indomethacin- inhibitable fraction is mediated by epinephrine-stimulated prostanoid production or release.

Alpha-2 adrenoceptor subtype causing nitric oxide-mediated vascular relaxation in rats.

Bockman C.S.; Gonzalez-Cabrera I.; Abel P.W.

Dr. P.W. Abel, Department of Pharmacology, Crisa III, Creighton Univ. School of Medicine, 2500 California Plaza, Omaha, NE 68178 USA

Journal of Pharmacology and Experimental Therapeutics (USA), 1996, 278/3 (1235-1243)

The alpha-2 adrenoceptor subtype and its signal transduction pathway mediating vascular relaxation in rats were studied in vitro using rings of superior mesenteric arteries. Removal of endothelium or incubation with N(G)- nitro-L-arginine completely blocked relaxant responses to UK14,304, suggesting endothelium-derived nitric oxide mediates relaxation. The order of potency for full (F) or partial (P) agonists causing relaxation was guanabenz (P) > UK14,304 (F) > clonidine (P) > epinephrine (F) > norepinephrine (F). Affinities (K(B)) of alpha-2 adrenoceptor subtype-selective drugs for blocking relaxation were obtained in side-by-side experiments comparing rat mesenteric arteries with pig coronary arteries. Relaxation of pig coronary arteries is known to be mediated by the alpha-2A adrenoceptor subtype. K(B) values in nM for rauwolscine (19), WB-4101 (265), SKF-104078 (197), spiroxatrine (128), and prazosin (1531) for blocking relaxation in rat arteries were consistent with their affinities for binding at the alpha-2D adrenoceptor subtype. K(B) values for rauwolscine and WB-4101, drugs distinguishing the alpha-2D from the alpha-2A adrenoceptor subtype, were significantly higher in blocking relaxation of rat arteries compared with pig arteries, suggesting the alpha-2D adrenoceptor subtype mediates NO-induced relaxation in rat arteries. We used forskolin to oppose alpha-2 adrenoceptor- mediated inhibition of cAMP formation by directly stimulating cAMP formation in endothelium. Forskolin did not affect the relaxant response to UK14,304, suggesting that cAMP is not involved in the coupling of alpha-2 adrenoceptors to nitric oxide-induced vascular relaxation.

Neurally mediated cardiac effects of forskolin in conscious dogs.

Iwase M.; Ishikawa Y.; Shen Y.-T.; Shannon R.P.; Sato N.; Ganguly P.K.; Eki T.; Vatner D.E.; Vatner S.F.

New England Regional Primate Center, One Pine Hill Dr., Southborough, MA 01772-9102 USA

American Journal of Physiology - Heart and Circulatory Physiology (USA), 1996, 271/4 40-4 (H1473-H1482)

Because major cardiovascular disease states are characterized by defects in adenylyl cyclase regulation, it becomes important to understand the mechanisms by which adenylyl cyclase activators affect inotropy and chronotropy in intact conscious animals. Accordingly, we examined the inotropic and chronotropic responses to forskolin in 11 normal conscious, chronically instrumented dogs and 3 dogs with ventricular denervation (VD). Left ventricular first derivative of pressure (LV dP/dt) increased by 96 plus or minus 7%, P < 0.05, in response to forskolin (50 nmol . kg-1 . min-1) in normal dogs and by significantly less, 52 plus or minus 14%, in VD dogs. Circulating norepinephrine (NE) levels increased similarly in both groups (from 226 plus or minus 18 to 389 plus or minus 33 pg/ml in normal dogs, from 177 plus or minus 23 to 329 plus or minus 71 pg/ml in VD dogs). In the presence of ganglionic blockade, the increase in LV dP/dt in response to forskolin was reduced (+62 plus or minus 4%) in normal dogs but was unchanged in VD dogs (+52 plus or minus 12%). Ganglionic blockade abolished the increase in circulating NE levels in both groups. Increases in heart rate in the presence of ganglionic blockade (+54 plus or minus 6 beats/min) were less than in the presence of atropine alone (+92 plus or minus 10 beats/min). Notably, the LV dP/dt and heart rate responses to forskolin were further attenuated by beta-adrenergic receptor blockade in the presence and absence of ganglionic blockade. Morphine also attenuated the increases in both LV dP/dt and plasma NE in response to forskolin. Increases in LV dP/dt in response to NKH-477 (30 microg/kg), a water-soluble forskolin derivative, were similar before and after ganglionic blockade (+63 plus or minus 8 and +51 plus or minus 10%, respectively). However, in vitro experiments in LV sarcolemmal membrane preparations demonstrated that stimulation of adenylyl cyclase by forskolin and NKH-477 was not affected by beta-adrenergic receptor blockade. These results indicate that in conscious dogs, inotropic and chronotropic effects of forskolin are not only due to direct activation of adenylyl cyclase, but the effects also are mediated by neural mechanisms and potentiated by the prevailing level of sympathetic tone.

Relaxant effects of forskolin on guinea pig tracheal smooth muscle

LUNG (GERMANY, WEST), 1987, 165/4 (225-237)

We investigated the relaxant effects of forskolin, a diterpene derivative isolated from the roots of Coleus forskohlii, on guinea pig airway smooth muscle by measuring the isometric tension of tracheal smooth muscle in vitro and transcutaneous POsub 2 during the histamine inhalation test (HIT) in vivo. Forskolin (10sup -sup 9-10sup -sup 5 M) caused dose-dependent relaxant effects on resting tone and on leukotriene Csub 4 (10sup -sup 7 M)-, leukotriene Dsub 4 (10sup -sup 7 M)-, and carbachol (3 x 10sup -sup 6 M)-induced contraction of tracheal smooth muscle. Moreover, with propranolol pretreatment the relaxant effect of forskolin on tracheal smooth muscle did not change, whereas with the same pretreatment the relaxant effect of isoproterenol diminished. Forskolin (10sup -sup 8-10sup -sup 6 M) raised tissue cyclic AMP levels dose-dependently in tracheal smooth muscle (6.7-359.9 pmol/mg protein). Forskolin (1 mg/kg) administered subcutaneously raised the respiratory threshold of RT-histamine in the HIT. The determination of the RT-histamine by measuring tcPOsub 2 was possible without anesthesia. These results suggest that forskolin relaxes airway smooth muscle in guinea pigs in vitro and in vivo by raising tissue cyclic AMP levels and that its actions are independent of beta-adrenoceptors.

Protective effect of forskolin against acetylcholine provocation in healthy volunteers - Comparison of two doses with fenoterol and placebo

WIEN. MED. WOCHENSCHR. (AUSTRIA), 1986, 136/23-24 (637-641)

Forskolin, a potent adenylate cyclase activating diterpene-derivative, isolated from the Indian plant Coleus forskohlii, was tested (double-blind and cross-over) in 12 healthy volunteers (nonsmokers) by whole body plethysmography. All drugs were administered by metered dose inhalers. The bronchodilating effect (after 5 minutes) was as good as following fenoterol. At the beginning (after 3 and 5 minutes) the protective effect against inhaled acetylcholine was as good as following fenoterol while later on (after 15 and 30 minutes) fenoterol resulted in a stronger action.

Autocrine role of interleukin 1beta in altered responsiveness of atopic asthmatic sensitized airway smooth muscle

Journal of Clinical Investigation (USA), 1997, 99/1 (117-124)

The role of IL-1beta in regulating altered airway responsiveness in the atopic/asthmatic sensitized state was examined in isolated rabbit tracheal smooth muscle (TSM) tissue and cultured cells passively sensitized with sera from atopic asthmatic patients or nonatopic/nonasthmatic (control) subjects. During half-maximal isometric contraction of the tissues with acetylcholine, relative to control TSM, the atopic sensitized TSM exhibited significant attenuation of both their maximal relaxation (P < 0.001) and sensitivity (i.e., -log dose producing 50% maximal relaxation) to isoproterenol and PGE2 (P < 0.05), whereas the relaxation responses to direct stimulation of adenylate cyclase with forskolin were similar in both tissue groups. The impaired relaxation responses to isoproterenol and PGE2 were ablated in sensitized TSM that were pretreated with either the IL-1 recombinant human receptor antagonist or an IL-1beta-neutralizing antibody. Moreover, extended studies demonstrated that, in contrast to their respective controls, both passively sensitized rabbit TSM tissue and cultured cells exhibited markedly induced expression of IL-1beta, mRNA at 6 h after exposure to the sensitizing serum, a finding similar to that also obtained in passively sensitized human bronchial smooth muscle tissue. Finally, unlike their respective controls, passively sensitized TSM tissue and cultured cells also displayed progressively enhanced release of IL-1beta protein into the culture media for up to 24 h after exposure to atopic/asthmatic serum. Collectively, these observations provide new evidence demonstrating that the altered responsiveness of atopic/asthmatic sensitized airway smooth muscle is largely attributed to its autologously induced expression and autocrine action of IL- 1beta.

Mechanism of cytokine-induced modulation of beta-adrenoceptor responsiveness in airway smooth muscle

Journal of Clinical Investigation (USA), 1996, 97/11 (2593-2600)

To elucidate the role of specific proinflammatory cytokines in regulating airway responsiveness, we examined the effects and mechanisms of action of IL-1beta, TNF-alpha, and IL-2 on the beta-adrenoceptor- and postreceptor-coupled transmembrane signaling mechanisms regulating relaxation in isolated rabbit tracheal smooth muscle (TSM) segments. During half-maximal isometric contraction of the tissues with acetylcholine, relaxation responses to isoproterenol, PGE2, and forskolin were separately compared in control (untreated) TSM and tissues incubated for 18 h with IL-1beta (10 ng/ml), TNF- alpha (100 ng/ml), or IL-2 (200 ng/ml). Relative to controls, IL-1beta- and TNF- alpha-treated TSM, but not IL-2-treated tissues, depicted significant attenuation of their maximal relaxation and sensitivity (i.e., -log dose producing 50% maximal relaxation) to isoproterenol (P < 0.001) and PGE2 (P < 0.05); whereas the relaxation responses to direct stimulation of adenylate cyclase with forskolin were similar in the control and cytokine-treated tissues. Further, the attenuated relaxation to isoproterenol and PGE2 was ablated in the IL-1beta-treated TSM that were pretreated with either the muscarinic M2-receptor antagonist, methoctramine (10-6 M), or pertussis toxin (100 ng/ml). Moreover, Western immunoblot analysis demonstrated that: (a) G(i) protein expression was significantly enhanced in membrane fractions isolated from IL-1beta-treated TSM; and (b) the latter was largely attributed to induced enhanced expression of the G(i)alpha2 and G(i)alpha3 subunits. Collectively, these observations provide new evidence demonstrating that IL- 1beta and TNF-alpha induce impaired receptor-coupled airway relaxation in naive TSM, and that the latter effect is associated with increased muscarinic M2- receptor/G(i) protein-coupled expression and function.

Secretory non-pancreatic phospholipase A2 and cyclooxygenase-2 expression by tracheobronchial smooth muscle cells

European Journal of Biochemistry (Germany), 1996, 235/3 (557-563)

Lipid mediators of inflammation, contribute to airway hyper-reactivity in asthma. Since production of lipid mediators is largely regulated by phospholipase A2 (PLA2), and since PLA2 expression in mesenchymal cells is induced by cytokines and other signals, we examined PLA2 expression by rat tracheobronchial smooth muscle cells (TBSMC). PLA2 expression in TBSMC cultures was markedly increased by tumour-necrosis factor (TNF)alpha (130-fold) and interleukin-1beta (IL-1beta, (7.4-fold). Lipopolysaccharide (LPS; 100 ng/ml) resulted in a 51-fold increase in extracellular PLA2 activity. PLA2 expression by LPS-stimulated or cytokine-stimulated cells was downregulated by dexamethasone. Whereas forskolin or dibutyrl cAMP increased PLA2 activity, inhibition of protein kinase A but not tyrosine kinase reduced PLA2 expression. Northern blot analysis showed that TNFalpha and IL-1beta increased both PLA2 and inducible cyclooxygenase (Cox-2) mRNA transcription. Addition of dexamethasone substantially blunted the increase in PLA2 and Cox-2 mRNA. In contrast, the level of Cox-1 mRNA was very low and did not change with the various treatments. Since proinflammatory lipid mediators have been implicated in the pathogenesis of asthma and PLA2 activity regulates generation of these lipid mediators, cytokine-stimulated synthesis and release of PLA2 by airway smooth cells may contribute to the potentiation of airway inflammation in asthma.

Evidence on the participation of the 3',5'-cyclic AMP pathway in the non-genomic action of 1,25-dihydroxy-vitamin D3 in cardiac muscle.

Mol Cell Endocrinol (NETHERLANDS) Dec 1991, 82 (2-3) p229-35

Several studies have suggested that vitamin D plays a role in cardiovascular function. It has been recently shown that in vitro treatment of vitamin D-deficient chick cardiac muscle with physiological concentrations of 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3) induces a rapid (1-10 min) increase of tissue 45Ca uptake which can be suppressed by Ca channel blockers. The hormone simultaneously stimulated heart microsomal membrane protein phosphorylation. Experiments were performed to investigate the existence of a relationship between these changes and to obtain information about the mechanism involved in 1,25(OH)2D3-induced modifications in cardiac protein phosphorylation. Dibutyryl cyclic AMP (10 microM) and forskolin (10 microM), known activators of the cAMP pathway, produced time courses of changes in 45Ca uptake by chick heart tissue similar to 1,25(OH)2D3 (10(-10) M). Analogously to the hormone, the effects of both compounds were abolished by nifedipine (30 microM) and verapamil (10 microM). In agreement with these observations, 1,25(OH)2D3 significantly increased (34-70%) heart muscle cAMP levels within 1-10 min of treatment. In addition, 1,25(OH)2D3 and forskolin caused similar changes in cardiac microsomal membrane protein phosphorylation (e.g. stimulation in 43 kDa and 55 kDa proteins). These changes were also evidenced by direct exposure of isolated heart microsomes to 1,25(OH)2D3, suggesting a direct membrane action of the hormone. The fast effects of 1,25(OH)2D3 on dihydropyridine-sensitive cardiac muscle Ca uptake could be reproduced in primary-cultured myocytes isolated from chick embryonic heart. Furthermore, the effects of the hormone could be suppressed by a specific protein kinase A inhibitor. These results suggest that 1,25(OH)2D3 affects heart cell calcium metabolism through regulation of Ca channel activity mediated by the cAMP pathway.

Effect of flosequinan on ischaemia-induced arrhythmias and on ventricular cyclic nucleotide content in the anaesthetized rat.

Br J Pharmacol (ENGLAND) Apr 1993, 108 (4) p1111-6

1. Flosequinan, milrinone, isoprenaline and forskolin given intravenously at similarly hypotensive doses have been evaluated in separate studies for their effect on ischaemia-induced arrhythmias and on ventricular cyclic nucleotide content following coronary artery ligation in the pentobarbitone anaesthetized rat. 2. Flosequinan did not affect mortality or arrhythmias following coronary artery ligation in either study and no change in ventricular cyclic nucleotide content was observed. 3. Isoprenaline caused a significant increase in mortality (P < 0.05) in both studies whereas milrinone and forskolin caused a significant increase in mortality in only one of the two studies conducted. All three agents caused significant increases in cyclic AMP which were associated with increased incidence of arrhythmias. 4. When compared at similarly hypotensive doses, flosequinan, in contrast to milrinone, isoprenaline and forskolin, did not influence ischaemia-induced arrhythmias or raise ventricular cyclic nucleotide levels in the anesthetized rat.

Arrhythmogenic effect of forskolin in the isolated perfused rat heart: Influence of nifedipine reduction of external calcium

CLIN. EXP. PHARMACOL. PHYSIOL. (Australia), 1989, 16/10 (751-757)

This study investigated first the effects of forskolin on cardiac rhythm, and second the roles of calcium in cardiac arrhythmogenesis by cAMP. Two series of experiments were performed. In the first series, forskolin was administered into the isolated perfused rat heart. In the second series, forskolin administration was preceded by administration of nifedipine, a calcium channel blocker, or infusion of a low concentration calcium solution. In both experiments, the myocardial cAMP level and electrocardiogram were determined. It was found that forskolin increased cAMP level as well as inducing arrhythmia. Pretreatment with nifedipine or a reduction of external calcium, that either maintained or further enhanced the forskolin-induced increase in the cAMP level, abolished the forskolin-induced arrhythmia. The results of the present study support the hypothesis that myocardial cAMP mediates cardiac arrhythmia, and provide evidence that calcium is essential in arrhythmia mediated by cAMP.

Hormone secretagogues increase cytosolic calcium by increasing cAMP in corticotropin-secreting cells

PROC. NATL ACAD. SCI. U. S. A. (USA), 1985, 82/23 (8034-8038)

Corticotropin (ACTH)-releasing factor, vasoactive intestinal peptide, and catecholamines - hormones that stimulate ACTH secretion and cAMP generation - increased cytosolic calcium in AtT-20 cells. The increase in intracellular calcium is presumably a consequence of the stimulated cAMP synthesis, since forskolin, an activator of the catalytic unit of adenylate cyclase, and the cAMP analog 8-bromoadenosine 3',5'-cyclic monophosphate (8Br-cAMP) also increased the cytosolic levels of this ion. Pretreatment with somatostatin, a neuropeptide that inhibits stimulation of the adenylate cyclase system and the secretion of ACTH blocked the increase of cytosolic calcium. The effect of 8Br-cAMP, which bypasses the cyclase, was not inhibited by somatostatin pretreatment. The source of the increased calcium appears to be mainly extracellular. This is indicated by the inability of the secretagogues to increase cytosolic calcium in a medium deprived of this ion or in the presence of blockers of voltage-gated calcium channels. The involvement of calcium channels in the calcium rise evoked by the secretagogues was supported by experiments using the whole-cell patch-clamp technique. In these experiments 8Br-cAMP increased voltage-dependent calcium currents. These results suggest the following chain of events in the receptor-mediated elevation of cytosolic calcium and the concomitant release of ACTH from AtT-20 cells: hormone-receptor binding > or = cAMP synthesis > or = protein kinase activation > or = calcium channel activation > or = increase in cytosolic calcium > or = many steps > or = ACTH release. Phorbol myristate acetate, a compound which does not stimulate cAMP generation but enhances the release of ACTH in AtT-20 cells, decreased the cytosolic calcium level.

The genesis of arrhythmias during myocardial ischemia. Dissociation between changes in cyclic adenosine monophosphate and electrical instability in the rat

CIRC. RES. (USA), 1985, 57/5 (668-675)

It has been proposed that increases in tissue cyclic adenosine monophosphate during ischemia may be responsible for the induction of arrhythmias that occur during the early minutes of ischemia. We have tested this hypothesis using the isolated perfused rat heart with coronary artery occlusion for 30 minutes. In control hearts, after a transient small rise, cyclic adenosine monophosphate content remained close to its preischemic value (3.0 + or - 0.1 nM/g dry weight) throughout the period of occlusion. Eight percent (1/12) of the hearts fibrillated. Ninety-two percent (11/12) of the hearts exhibited ventricular tachycardia, and the mean total number of premature ventricular complexes was 528 + or - 121. Inclusion of epinephrine (1.0 muM) in the perfusion fluid elevated cyclic adenosine monophosphate prior to coronary occlusion (to 10.7 + or - 0.6 nM/g dry weight) and also throughout the ischemic period. It also increased arrhythmias such that 83% (20/24) of hearts fibrillated, 100% exhibited ventricular tachycardia, and the mean number of premature ventricular complexes increased to 747 + or - 86. Inclusion of forskolin (0.2 muM), which stimulates adenyl cyclase independently of the beta-receptor, increased cyclic adenosine monophosphate content to a greater extent than epinephrine, to 14.1 + or - 0.9 nM/g dry weight before the onset of ischemia and to 8.2 + or - 0.4 nM/g dry weight after 30 minutes of ischemia. Despite the large increases in cyclic adenosine monophosphate, there was no increase in rhythm disturbances which were less than those seen in controls. Thus, no hearts fibrillated, the incidence of ventricular tachycardia was reduced to 58% (7/12), and the mean number of premature ventricular complexes was greatly reduced (79 + or - 29, P<0.001 compared to the number with drug carrier alone). Higher concentrations of both epinephrine and forskolin caused changes that were qualitatively similar to those seen with the lower concentrations. In addition, when hearts were paced at 400 impulses/min, again only epinephrine increased the severity of ischemia-induced arrhythmias. In conclusion, despite its ability to increase cyclic adenosine monophosphate content to a greater extent than epinephrine, forskolin exerts an antiarrhythmic effect. This suggests that increased cyclic adenosine monophosphate content is not necessarily involved in the genesis of ischemia-induced arrhythmias, and that some other facet of adrenoceptor stimulation or catecholamine action may be involved.

Effects of high K on relaxation produced by drugs in the guinea-pig tracheal muscle

RESPIR. PHYSIOL. (NETHERLANDS), 1985, 61/1 (43-55)

In the guinea-pig tracheal smooth muscle, effects of various relaxants were compared in normal (5.9 mM) and excess (40 mM) K media. The relaxing efect of calcium-channel blockers, nifedipine and verapamil (group I) was potentiated by increasing the external K concentration. The effect of the drugs which are supposed to increase intracellular cyclic AMP, such as isoprenaline, forskolin, isobutylmethylxanthine, theophylline, dibutyryl cyclic AMP (group II) was moderately reduced by excess K. Nitroprusside, 8-bromo-cyclic GMP and sodium nitrite (group III) are generally considered to increase intracellular cyclic GMP and their effect was markedly reduced by excess K. When the tension development was made the same at 5.9 mM K and 40 mM K by adjusting the Ca concentration, the relaxing effect was similar and independent of the K concentration both for group II and group III drugs. It seems that the group II drugs can better overcome a large influx of Ca than group III drugs.

Forskolin inhibits ouabain-sensitive ATPase in the medulla of rat kidney

IRCS MED. SCI. (ENGLAND), 1983, 11/11 (957-958)

The diterpene forskolin, a cardiotonic, vasodilatory and hypotensive drug, is a potent activator of adenylate cyclase but little is known about its effects on other membrane bound enzymes. Total ATPase, in the absence of ouabain, and ouabain-insensitive ATPase, in the presence of 1 mM ouabain, were measured by the enzymatic technique of Fritz and Hamrick. The difference between total and ouabain-insensitive ATPase activity is referred to as Na+Ksup +-ATPase. The protein content was determined according to Lowry. In cortex homogenates, no significant modification of total, ouabain-insensitive and Nasup +Ksup +-ATPase activities occurred in the presence of 10sup -sup 4 M forskolin. In medulla homogenates, forskolin (10sup -sup 4 M) caused a significant 55% decrease of Nasup +Ksup +-ATPase activity. The inhibition is dose-dependent but not complete at 10sup -sup 4 M forskolin, higher concentrations of the drug could, however, not be prepared because of its limited solubility. It would be interesting to correlate this result with a physiological difference of the cortical and medullary Nasup +Ksup +-ATPase.