Protein kinase A-dependent and -independent effects of isoproterenol in rat isolated mesenteric artery: interactions with levcromakalim

The effect of beta-adrenoceptor activation on levcromakalim-induced relaxation was investigated in myograph-mounted rat mesenteric arteries. The nonselective beta-adrenoceptor agonist isoproterenol (at a concentration causing approximately 30% relaxation of methoxamine-induced tone) potentiated relaxation to levcromakalim; higher concentrations exerted no additional effect. The modulatory and relaxant effects of isoproterenol were inhibited by the beta(1)-adrenoceptor antagonist atenolol, but the ATP-sensitive K(+) (K(ATP)) channel inhibitor glibenclamide did not inhibit relaxations to isoproterenol. The protein kinase A inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate triethylamine (Rp-cAMPS) inhibited the ability of isoproterenol to modulate levcromakalim relaxation. However, neither Rp-cAMPS nor N-[2-(p-bromocinnamylamino)ethyl]-6-isoquinolinesulfonamide (H-89) (another protein kinase A inhibitor) markedly reduced isoproterenol-induced relaxation, although Rp-cAMPS inhibited relaxations induced by forskolin (an adenylyl cyclase activator). Iberiotoxin (50 nM), an inhibitor of large conductance Ca(2+)-activated K(+) channels (BK(Ca)), attenuated isoproterenol relaxation. Moreover, both Rp-cAMPS and H-89 caused inhibition of the effects of isoproterenol in the presence of iberiotoxin, whereas glibenclamide did not. We conclude that isoproterenol modulates the actions of levcromakalim through beta(1)-adrenoceptors and protein kinase A, even though K(ATP) channels do not contribute to its relaxant effects. However, the major relaxant mechanism for isoproterenol appears to be protein kinase A-independent activation of BK(Ca), with cyclic AMP-dependent mechanisms only being unmasked when the BK(Ca) mechanism is inhibited. Although direct G protein-mediated activation of BK(Ca) has been demonstrated previously in electrophysiological studies of single smooth muscle cells, this is the first time that such a mechanism has been shown to be functionally important in an intact blood vessel preparation.     

Ethnic differences in cyclic AMP accumulation: effect on alpha 2, beta 2, and prostanoid receptor responses

Platelet alpha 2- and lymphocyte beta 2-adrenergic receptor density and responsiveness and platelet responses to prostaglandin E2 (PGE2) were measured in black subjects and white subjects. Mean (+/- SEM) alpha-receptor density was greater in white subjects (248 +/- 29 versus 392 +/- 31 fmol/mg protein; p less than 0.005), whereas beta-receptor densities were similar (black subjects, 19.2 +/- 2.2 fmol/mg protein; white subjects, 15.2 +/- 1.4 fmol/mg protein). Basal platelet cyclic AMP levels (black subjects, 7.8 +/- 1.1 pmol/mg protein; white subjects, 14.5 +/- 2.4 pmol/mg protein; p less than 0.05) and PGE2-induced increases in platelet cyclic AMP levels were higher in white subjects (black subjects, 47.1 +/- 3.3 pmol/mg protein; white subjects, 65.5 +/- 3.4 pmol/mg protein; p less than 0.001). Basal lymphocyte cyclic AMP levels (black subjects, 1.2 +/- 0.3 pmol/10(6) cells; white subjects, 2.8 +/- 0.6 pmol/10(6) cells; p less than 0.05) and mean isoproterenol-induced cyclic AMP increases were also higher in white subjects (black subjects, 6.2 +/- 0.6 pmol/10(6) cells; white subjects, 8.5 +/- 0.8 pmol/10(6) cells; p less than 0.05). Responses to isoproterenol as fold stimulation of basal were not significantly higher in black subjects (8.51 +/- 0.97 versus 6.16 +/- 0.86 fold-stimulation of basal). Platelet responses to PGE2 as fold stimulation of basal (black subjects, 7.9% +/- 0.7%; white subjects, 8% +/- 1.3%) and alpha 2-receptor-mediated inhibition of PGE2 responses by UK-14304 (black subjects, 38.2% +/- 1.7%; white subjects, 40.6% +/- 2.3%) were also similar in the two groups.     

Effects of forskolin on contractility and cyclic AMP levels in rabbit detrusor muscle

Forskolin caused a concentration-dependent relaxation and increase in cyclic AMP levels in rabbit detrusor muscle. Propranolol, a beta-adrenoceptor antagonist, did not affect the relaxation induced by forskolin. 3-isobutyl-1-metylxanthine (IBMX), a cyclic nucleotide phosphodiesterase inhibitor, potentiated the relaxation induced by forskolin. These data suggest that the relaxation of rabbit detrusor muscle induced by forskolin is mediated by cyclic AMP accumulation resulting from activation of adenylate cyclase.     

The time course of changes in force and cyclic AMP levels produced by isoproterenol and forskolin in isolated rabbit detrusor muscle

The effects of forskolin and isoproterenol on contractile force and cyclic AMP (cAMP) levels were compared in rabbit detrusor. Both forskolin and isoproterenol produced relaxation of rabbit detrusor and an increase in cAMP levels in the tissue. Although the relaxant response to forskolin was similar to that of isoproterenol, the increase in cAMP levels induced by forskolin was significantly larger than that induced by isoproterenol. These results suggest that there is a discrepancy in the relaxation responses and cAMP levels in response to forskolin and isoproterenol.     

Inhibition of adenylate cyclase attenuates adenosine receptor-mediated relaxation in coronary artery.

This study was designed to evaluate whether the adenylate cyclase inhibitor 2',5'-dideoxyadenosine (DDA) would attenuate the relaxation produced by adenosine analogs in order to provide functional evidence in support of the working hypothesis that adenosine receptor-mediated relaxation of coronary artery involves adenylate cyclase. Rings from porcine left anterior descending coronary artery were mounted in organ chambers for measurement of isometric force. Rings contracted with KCl (30 mM) relaxed in a concentration-dependent manner to 2-chloroadenosine (CAD), 5'-N-ethylcarboxamidoadenosine (NECA), isoproterenol, sodium nitroprusside (SNP) and forskolin. Treatment of coronary rings with DDA (50 microM) significantly attenuated the relaxation produced by CAD, NECA, forskolin and isoproterenol, but had no effect on the relaxation response to SNP. The nucleoside transport inhibitor dilazep (10 microM) completely reversed the inhibitory effect of DDA on the relaxation produced by forskolin and CAD, whereas dilazep only partially reversed the DDA inhibition of NECA-induced relaxation. In a membrane preparation from porcine coronary artery CAD, but not NECA, increased cyclic AMP production in a GTP-dependent manner. DDA significantly decreased basal cyclic AMP production and also decreased CAD-, forskolin-, GTP- and NaF-stimulated cyclic AMP production. These results provide functional and biochemical evidence in support of the working hypothesis that adenosine receptor-mediated coronary relaxation involves adenylate cyclase. Furthermore, the results from this study suggest that the signaling mechanisms responsible for adenosine receptor-mediated coronary relaxation are more complicated than a single receptor coupled with adenylate cyclase because 1) dilazep completely reversed the inhibitory effect of DDA on the CAD relaxation but not the NECA relaxation, and 2) NECA did not increase cyclic AMP production.     

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

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) Gi 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 Gi alpha2 and Gi alpha3 subunits. Collectively, these observations provide new evidence demonstrating that IL-lbeta and TNF-alpha induce impaired receptor-coupled airway relaxation in naive TSM, and that the latter effect is associated with increased muscarinic M2-receptor/Gi protein-coupled expression and function.     

Interaction of prostaglandins with adenosine diphosphate induced increase in cytosolic free calcium in human platelets.

The interaction of prostaglandins with changes in cytosolic Ca2+ concentration ([Ca2+]) and aggregation of human platelets induced by adenosine diphosphate (ADP) were investigated. Cytosolic [Ca2+] was measured with the fluorescent dye Quin2. Addition of ADP (0.25-2.5 mumol l-1) to platelet suspensions produced a dose dependent increase in cytosolic [Ca2+] from a basal level of 51 +/- 1 nmol l-1 to maximum levels exceeding 1 mumol l-1 and induced platelet aggregation. Chelation of extracellular calcium with 100 mumol l-1 EGTA markedly reduced the increase in cytosolic [Ca2+] induced by 0.25 mumol l-1 ADP, while pretreatment with the calcium entry blocker verapamil was without effect. Stimulation of cyclic AMP with prostaglandins (PGD2, PGE1, PGE2, PGI2, but not PGF2 alpha) and forskolin, or incubation with dibutyryl-cAMP, inhibited the rise in cytosolic [Ca2+] and platelet aggregation following ADP. We conclude that prostaglandins inhibit the increase in cytosolic [Ca2+] and aggregation of human platelets induced by ADP, probably by stimulation of cyclic AMP generation, thereby opposing the mechanism by which ADP increases cytosolic [Ca2+] and subsequently induces platelet aggregation.     

Impaired beta adrenergic receptor binding and function in cystic fibrosis neutrophils.

Cystic fibrosis (CF), a genetic disease characterized by abnormalities of exocrine gland and mucociliary function, has recently been shown to be associated with abnormal adrenergic and cholinergic physiologic responses in addition to decreased beta adrenergic-induced cyclic AMP generation in human leukocytes. In this study we have attempted to elucidate the nature of this hyporesponsiveness by assessing beta adrenergic receptor number and affinity (KD) in the intact neutrophil using the antagonist ligand [3H] dihydroalprenolol and cyclic AMP responses to isoproterenol in addition to histamine, and prostaglandin E1 in CF subjects, CF obligate heterozygotes (CFH), and normal control subjects. CF patients had significantly less (p less than 0.025) cyclic AMP stimulation above basals levels with isoproterenol (0.1 microM to 0.1 mM), compared with control values, but no consistent differences between groups were noted with histamine or PGE1. CF neutrophils had significantly fewer (p less than 0.005) beta adrenergic receptors per neutrophil (398.0 +/- 54.2 vs. 819.4 +/- 67.2) compared with control neutrophils, but the KD (0.740 +/- 0.11 vs. 0.630 +/- 0.05 nM) did not differ significantly (p greater than 0.05). There was no correlation between clinical severity and either cyclic AMP generation or dihydroalprenolol binding (r = 0.27 and 0.24, respectively, p greater than 0.05). The CFH group had approximately 50% of the cyclic AMP stimulation compared with controls, but the number (909.8 +/- 89.3) and KD (0.710 +/- 0.09 nM) of their beta adrenergic receptors were indistinguishable from control subjects. These findings suggest "down regulation" of the beta receptor in the CF patient. The cause of this remains unknown. Although the etiology of the decreased cyclic AMP responses in CFH was not due to decreased beta adrenergic receptors as assessed by antagonist ligand binding, further studies inthe CFH group to include agonist binding, receptor-adenylate cyclase coupling, intrinsic adenylate cyclase activity, and catecholamine metabolism may help determine the basic cause of beta adrenergic hyperesposiveness in both CFH and CF.     

Relaxant effects of beta-carbolines on rat aortic rings.

The effects of two beta-carbolines, methyl 6,7-dimethoxy-4-ethyl-beta- carboline-3-carboxylate (DMCM) and ethyl beta-carboline-3-carboxylate (beta CCE) were assayed on rat aortic rings precontracted with different agonists. The beta-carbolines tested induced a concentration-dependent (2-200 microM) relaxation of aortic rings precontracted with 30 mM KCl. This relaxation was not modified by the removal of the rat aortic endothelium. Contractions elicited by the activation of either voltage-gated calcium channels (0.05 microM BAY K 8644) or receptor-operated calcium channels (0.1 microM norepinephrine), as well as contractions produced by the entry of calcium as a lipid-soluble complex (10 microM A23187), were also reduced by DMCM and by beta CCE. In addition, whereas DMCM did not modify calmodulin activity, both beta-carbolines inhibited in a concentration-dependent manner (0.6-200 microM) the rat aortic cyclic nucleotide phosphodiesterase activity. Moreover, DMCM as well as beta CCE potentiated the relaxation of K(+)-contracted aortic rings induced by the stimulation of either adenylyl cyclase with forskolin (0.1-1 microM) or guanylyl cyclase with sodium nitroprusside (0.1-100 nM). The intracellular rat aortic levels of cyclic AMP measured in the presence of 0.1 microM forskolin were increased by 100% in the presence of DMCM. On the other hand, 6 microM DMCM potentiated the relaxation induced by nifedipine in K(+)-contracted aortic rings, whereas the K+ channel blocker 10 mM tetraethylammonium did not modify the relaxation elicited by DMCM in the norepinephrine-contracted preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostaglandin E2 inhibition of secretagogue-stimulated [14C]aminopyrine accumulation in rat parietal cells: a model for its mechanism of action

Prostaglandin E2 (PGE2) differentially inhibited histamine and isoproterenol stimulation of [14C]aminopyrine accumulation in rat parietal cell preparations. Low concentrations of PGE2 decreased the maximum response to isoproterenol whereas higher concentrations increased the EC50 of histamine with only a modest effect on the maximum response. Also, PGE2 potentiated dibutyryl cyclic AMP stimulation of aminopyrine accumulation in either the absence or presence of carbachol. In contrast, PGE2 inhibited potentiation between carbachol and histamine due to its inhibitory effect on histamine and possibly also to an inhibitory effect on cholinergic activity. Islet activating protein prevented the inhibitory actions of PGE2. To account for these results a model is presented based on the recent proposal by Gilman (Cell 36: 577-579, 1984) of an interaction between components of adenylyl cyclase stimulatory and inhibitory guanine nucleotide binding proteins.     

Effects of pimobendan, a cardiotonic and vasodilating agent with phosphodiesterase inhibiting properties, on isolated arteries and veins of rats.

Effects of pimobendan (PBD) were investigated on isolated rat blood vessels. PBD dose-dependently relaxed aortic, femoral arterial and mesenteric venous preparations precontracted with KCl and reduced the amplitude of spontaneous contractions of portal venous preparations; the sensitivity to PBD was femoral greater than portal greater than mesenteric greater than aorta. Relaxation response of the femoral artery to PBD was not changed by propranolol and aminophylline. Glyceryl trinitrate (GTN), isoproterenol (ISO), forskolin and adenosine also elicited dose-dependent relaxations of femoral arteries; the rank order of potency (mean negative log EC50 value) was GTN greater than ISO greater than PBD = forskolin greater than adenosine. The relaxation responses to PBD and isobutyl methylaxanthine (IBMX) were not attenuated with removal of endothelial cells. In the femoral artery, methylene blue diminished GTN-induced relaxation but not PBD-induced relaxation. PBD and IBMX increased the relaxation responses of the artery to cyclic AMP-forming drugs (ISO, forskolin and adenosine) but not a cyclic GMP-forming drug (GTN). PBD and IBMX increased the relaxation response of mesenteric veins to ISO. The drugs noncompetitively inhibited arterial contractions accompanied by voltage-dependent and alpha-adrenoceptor-operated Ca2+ influxes. In the absence of extracellular Ca2+, PBD and IBMX reduced contractile responses of arteries to norepinephrine but not caffeine. The present results suggested that PBD relaxed the blood vessels, at least in part, through an intracellular accumulation of cyclic AMP.     

Activation of phospholipase D by E-series prostaglandins in human erythroleukemia cells.

Phospholipase D (PLD) can be activated by a variety of receptor agonists in different cell types. However, an effect of prostaglandins (PGs) on the activity of this enzyme has not been demonstrated previously. In this study, we found that PGE1 could stimulate PLD in human erythroleukemia cells, as measured by phosphatidylethanol formation, with an ED50 of 3.5 x 10(-7) M. PGE2 was also active, but other PGs including prostacyclin, PGD2 and PGF2, had no effect. PGE1 also elicited cyclic AMP production over the same concentration range that activated PLD. However, it is unlikely that cyclic AMP per se is responsible for PGE-induced PLD activation, because PLD could be substantially activated by PGE2 at concentrations (0.1-1 microM) which did not stimulate cyclic AMP production. Furthermore, no increase of phosphatidylethanol formation could be observed when cells were treated with other adenylyl cyclase-activating agents such as prostacyclin, forskolin and vasoactive intestinal peptide. In Ca+(+)-free medium, PLD activation by PGE1 and PGE2 was greatly reduced, indicating that their effect was through a Ca+(+)-dependent pathway. Pretreatment of cells with pertussis toxin abolished PGE1- and PGE2-stimulated PLD activity, implying the involvement of a G protein in the PGE-mediated signal transduction pathway. Our results not only indicate that E-series PGs may initiate some of their cellular effects through a novel pathway, activation of PLD, but also suggest that PGE-stimulated PLD activity in human erythroleukemia cells is Ca+(+)-dependent and is regulated via a pertussis toxin-sensitive G protein.     

Aspirin potentiates prestimulated acid secretion and mobilizes intracellular calcium in rabbit parietal cells.

The effects of aspirin on gastric acid secretion were studied in isolated rabbit parietal cells (PC). Aspirin (10(-5) M) potentiated histamine-, dibutyryl cyclic AMP (dbcAMP)-, forskolin- and 3-isobutyl-1-methylxanthine-stimulated acid secretion without affecting basal acid secretion. Augmentation of secretagogue-stimulated acid secretion by aspirin was dependent on calcium (Ca2+) since potentiation was blocked by removal of extracellular Ca2+ ([Ca2+]o) or addition of the calcium antagonist lanthanum chloride. Using the Ca2+ probe fura-2, aspirin (10(-6) - 2 X 10(-5) M) rapidly increased intracellular free Ca2+ concentration ([Ca2+]i) in a dose-dependent manner. The source of released Ca2+ was intracellular as demonstrated by depletion of intracellular Ca2+ and [Ca2+]o with EGTA washing. Aspirin did not affect several other signal transduction sites involved in stimulus-secretion coupling, including the H2 receptor, intracellular cyclic AMP (cAMP), inositol 1,4,5, triphosphate (IP3) and H+,K(+)-ATPase. Aspirin decreased PC prostaglandin E2 (PGE2) content by 98%. Exogenous dimethyl PGE2 (dmPGE2) inhibited both histamine-stimulated acid secretion and its enhancement by aspirin. In contrast, dmPGE2 abolished aspirin-induced potentiation of dbcAMP-stimulated acid secretion by augmenting the dbcAMP-stimulated response. These results indicate that aspirin acts at a site beyond the adenylate cyclase/cAMP system and before the proton pump, presumably by releasing Ca2+ from an IP3-independent intracellular storage pool and by inhibiting PGE2 generation.     

Age-related decrease in beta adrenergic receptor-mediated vascular smooth muscle relaxation

Beta adrenergic receptor-mediated vascular smooth muscle relaxation decreases with increasing age. We have examined the mechanism responsible for this phenomenon using rat mesenteric arteries from young (5-6 weeks) and older (10-12 months) rats. The beta adrenergic agonist isoproterenol produced a dose-dependent relaxation of serotonin-constricted mesenteric artery rings from young rats, whereas the maximal ability of isoproterenol to relax arterial rings from the older rats was found to be reduced markedly (92.7 vs. 27.6%, P less than .0001). The relaxation responses caused by acetylcholine and nitroglycerin, which appear to act independently of cyclic AMP (cAMP), are similar in the two groups. The loss in responsiveness of the mesenteric artery to isoproterenol was not explained by a change in beta receptor number in the vessels (29 +/- 4 in young rats vs. 31 +/- 7 fmol/mg of protein in the older rats). The maximal stimulation of cAMP accumulation by isoproterenol was lower in the older vessels; forskolin activated cAMP accumulation equally in the two groups. However, the vessels from the older rats were less sensitive to forskolin-induced vascular relaxation. Also, the ability of dibutyryl cAMP to promote vascular relaxation was diminished in the older vessels. These data suggest that the diminished cAMP accumulation in older vessels in response to isoproterenol might not necessarily in itself explain completely the reduced physiological response and that an additional defect in the beta adrenergic-mediated relaxation in the vascular smooth muscle of older rats may lie at the level of cAMP-dependent protein kinase activation or more distally.     

Inhibitory effect of methacholine on drug-induced relaxation, cyclic AMP accumulation, and cyclic AMP-dependent protein kinase activation in canine tracheal smooth muscle

Functional antagonism between bronchoconstricting and bronchodilating pathways was examined in canine tracheal smooth muscle. Trachealis strips were contracted with either 0.3 microM (EC55) or 3.0 microM (EC80) methacholine before being relaxed by the cumulative addition of isoproterenol, prostaglandin E2, or forskolin. The EC50 for all three relaxants was increased 10-fold in tissues contracted with 3.0 microM methacholine vs. those contracted with 0.3 microM methacholine. Moreover, contracting tissues with the higher concentration of methacholine reduced the maximum relaxation induced by prostaglandin E2 and isoproterenol. Forskolin produced total relaxation regardless of the concentration of methacholine used and thus was a much more effective bronchodilator than either isoproterenol or prostaglandin E2. The inhibitory effect of methacholine on the relaxant response to these agents was paralleled by a reduction in drug-stimulated cyclic AMP-dependent protein kinase activity. Methacholine reduced the maximum activation of cyclic AMP-dependent protein kinase elicited by isoproterenol, prostaglandin E2 and submaximal concentrations of forskolin, which was a much more powerful enzyme activator than the other two agents. The ability of a maximum concentration of forskolin (30 microM) to activate cyclic AMP-dependent protein kinase was not inhibited by methacholine. Although methacholine also appeared to suppress drug-stimulated cyclic AMP accumulation, the inhibitory effect was only statistically significant in forskolin-treated tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms of impaired beta-adrenoceptor-induced airway relaxation by interleukin-1beta in vivo in the rat.

We studied the in vivo mechanism of beta-adrenergic receptor (beta-AR) hyporesponsiveness induced by intratracheal instillation of interleukin-1beta (IL-1beta, 500 U) in Brown-Norway rats. Tracheal and bronchial smooth muscle responses were measured under isometric conditions ex vivo. Contractile responses to electrical field stimulation and to carbachol were not altered, but maximal relaxation induced by isoproterenol (10(-6)-10(-5) M) was significantly reduced 24 h after IL-1beta treatment in tracheal tissues and to a lesser extent, in the main bronchi. Radioligand binding using [125I]iodocyanopindolol revealed a 32+/-7% reduction in beta-ARs in lung tissues from IL-1beta-treated rats, without any significant changes in beta2-AR mRNA level measured by Northern blot analysis. Autoradiographic studies also showed significant reduction in beta2-AR in the airways. Isoproterenol-stimulated cyclic AMP accumulation was reduced by IL-1beta at 24 h in trachea and lung tissues. Pertussis toxin reversed this hyporesponsiveness to isoproterenol but not to forskolin in lung tissues. Western blot analysis revealed an IL-1beta-induced increase in Gi(alpha) protein expression. Thus, IL-1beta induces an attenuation of beta-AR-induced airway relaxation through mechanisms involving a reduction in beta-ARs, an increase in Gi(alpha) subunit, and a defect in adenylyl cyclase activity.     

Role of cyclic AMP protein kinase in decreased arterial cyclic AMP responsiveness in hypertension

A decreased relaxation responsiveness to isoproterenol and forskolin is manifest in aortic smooth muscle isolated from spontaneously hypertensive rats (SHR) when compared with normotensive Wistar Kyoto (WKY) rats. Inasmuch as the effector of cyclic AMP (cAMP) is cAMP-dependent protein kinase, we sought to determine if alterations in this enzyme might be responsible for this decreased responsiveness to cAMP-increasing vasodilators. The concentration of cAMP protein kinase activity in aortic, carotid and caudal arteries (approximately 300 pmol/mg of protein per min) was similar in both WKY and SHR. Activity in femoral arteries from SHR and WKY rats was greater (approximately 600 pmol/mg/min); branches of the femoral artery from SHR had less protein kinase activity (660 pmol/mg/min) than their WKY counterparts (1000 pmol/mg/min). There were no differences between WKY and SHR in isozymic distribution of soluble cAMP protein kinase in any of these sources of arterial smooth muscle. Concentration and temporal-related relaxation of KCl-contracted aortic muscle strips by forskolin was associated with concomitant activation of cAMP protein kinase in both groups. The rate and extent of kinase activation was similar for both groups even though the rate and extent of relaxation was markedly less in SHR. These findings show that neither the concentration, isozymic distribution nor activation of cAMP-dependent protein kinase are different in aortic smooth muscle isolated from SHR when compared with WKY animals. Thus, decreased relaxation responsiveness to cAMP-increasing vasodilators is probably not related to events proximal to and including activation of arterial cAMP-dependent protein kinase.     

Relaxation of vascular smooth muscle by isoproterenol, dibutyryl-cyclic AMP and theophylline

There is evidence that the relaxation of vascular smooth muscle produced by isoproterenol or cyclic AMP is mediated by membrane hyperpolarization. The current study investigates the possibility that this hyperpolarization, and hence the relaxation, may be produced by activation of the electrogenic sodium pump. Rat and pig tail artery strips were placed in a 1.0-mM potassium solution for 15 min. This procedure results in a decrease in the activity of the sodium pump. The strips were then made to contract in response to norepinephrine. Two minutes later, the concentration of potassium was increased to 6.0 mM and a relaxation occurred. The amplitude of this relaxation reflects the activity of the sodium pump. Either isoproterenol or dibutyryl-cyclic AMP causes an enhancement (time or degree) of potassium-induced relaxation. Theophylline potentiated potassium-induced relaxation in pig arteries but not in rat arteries. The relaxant action of isoproterenol on 1.0 mM barium contractures of rat arteries was inhibited by treatment with ouabain or with potassium-free solution. Ouabain inhibited the relaxant action of isoproterenol in pig arteries contracted with depolarizing potassium solution but not in rat tail arteries. Dibutyryl-cyclic AMP, theophylline and nitroprusside caused relaxation of serotonin-induced contractions; however, in rat arteries these responses were not inhibited by ouabain or by the absence of potassium. Similar studies on tail arteries from baboons, dogs, pigs and cats showed that relaxation by dibutyryl-cyclic AMP or by theophylline had some dependency on the activity of the sodium pump. These observations are consistent with the following conclusions: 1) isoproterenol and cyclic AMP potentiate the electrogenic pumping of sodium and potassium responsible for potassium-induced relaxation; 2) the relaxing action of isoproterenol, dibutyryl-cyclic AMP and theophylline are dependent upon experimental conditions and the species from which the vascular tissue is obtained; and 3) there is a component of isoproterenol- and cyclic AMP-induced relaxation which is not altered by inhibition of the electrogenic sodium pump in the rat.     

Effect of isoproterenol and D-600 on calcium movements in rat myometrium.

The effects of two smooth muscle relaxants, isoproterenol and D-600, on calcium movements in rat myometrium were investigated. Both relaxants caused a nonspecific increase in 45-Ca efflux due to changes in mechanical tension but the additional net loss expected on the basis of previous studies with isoproterenol could not be demonstrated due to a high background of calcium exchange. Analysis of 45-Ca and 40-Ca residual in the muscle after efflux experiments and washing of the tissue in a Ca-deficient solution containing LaCl3 (2.0 mM) showed that the specific activity ratio 45-Ca/40-Ca was unaltered with isoproterenol and, thus, the net loss of calcium occurred equally from slowly and rapidly exchanging compartments. "Pulse label" experiments in which 45-Ca and either isoproterenol or D-600 were added simultaneously for a 2-minute period demonstrated that both relaxants decreased the 45-Ca space; however, the specific activity ratio 45-Ca/40-Ca in the tissue was reduced while 40-Ca remained unchanged in the presence of D-600. With isoproterenol, the 45-Ca/40-Ca ratio was increased while 40-Ca was reduced. These data support the hypothesis that isoproterenol stimulates a net efflux of calcium whereas D-600 inhibits the influx of calcium. Since previous studies have demonstrated that relaxants which increase cyclic3,5-adenosine monophosphate (cyclic AMP) (isoproterenol, dibutyryl cyclic AMP and papaverine) produce consistent decreases in tissue Ca but others (D-600) do not, it is concluded that relaxants which increase tissue cyclic AMP stimulate a net efflux of calcium but other antagonists may act by inhibiting calcium influx into rat myometrium.     

Stimulation of osmotic water flow in toad bladder by prostaglandin E1. Evidence for different compartments of cyclic AMP.

The effect of prostaglandin E1 (PGE1) on osmotic water flow across toad bladder and cyclic AMP content of the mucosal epithelial cells has been determined under basal conditions and in the presence of either theophylline or antidiuretic hormone (ADH); Under basal conditions and with PGE1 concentrations from 10(-8) to 10(-5) M no evidence of stimulation of water flow was observed, and with 10(-7) M PGE1 a significant inhibition was foundmcyclic AMP content under control conditions was 8 pmol/mg protein. It was 9 at 10(-8) M PGE1, 13 at 10(-7) M, 16 at 10(-6) M, and 23 at 10(-5) M. In the presence of theophylline, 10(-8) and 10(-7) M PGE1 inhibited the theophylline-induced water flow as expected. In contrast, 10(-6) and 10(-5) M PGE1 enhanced the rate of water flow. Theophylline increased cyclic AMP content from 8 to 18 pmol/mg protein. PGE1 in the presence of theophylline caused marked increases in cyclic AMP content; The content was 23 at 10(-7) M, 41 at 10(-6) M, and 130 at 10(-5) M; Thus PGE1 stimulates theophylline-induced water flow at cyclic AMP concentrations somewhere between 23 and 41 pmol/mg. Further evidence along these lines was obtained from experiments in which the effects of PGE1 on ADH-induced water flow were studied. Inhibitory effects of PGE1 were not observed at concentrations of PGE1 which raised the level of intracellular cyclic AMP to 30 pmol/mg protein or higher. These results were obtained despite the fact that all four concentrations of PGE1 tested were found capable of inhibiting ADH-induced water flow under appropriate conditions or, in other words, were inhibiting the adenylate cyclase controlling water flow, Thus the increase in cyclic AMP content in response to PGE1 is not derived from this enzyme. Thus the stimulation of water flow by PGE1 in the presence of theophylline is thought to be caused by cyclic AMP spilling over from one compartment to the water flow compartment. No evidence was obtained to directly suggest spillover into the sodium transport compartment. Furthermore evidence is discussed to suggest that most of the cyclic AMP generated in the tissue does not originate from the enzyme controlling sodium transport. As cyclic AMP-stimulated water flow and sodium transport are thought to occur in one cell type, the granular cells, distinct pools of cyclic AMP are thought to be present in one and the same cell type. Thus one pool controls water flow and one controls sodium transport. With high concentrations of PGE1 in the presence of theophylline or high concentrations of ADH, the adenylate cyclase responsible for water flow is inhibited; However, PGE1 can stimulate a tissue adenylate cyclase to sufficiently high levels that cyclic AMP spills over into the "water flow compartment" and thus stimulates water flow.