Effects of dopamine (DA) and SKF 82526, a selective DA1-receptor agonist, on vascular resistances in the canine forelimb

This study was performed to determine the presence of vascular dopamine (DA1) receptors in the canine forelimb. Local i.a. infusions of DA (2, 4 or 8 micrograms base/min) produced cutaneous and skeletal muscle vasoconstriction in the forelimb comparable to that produced by local i.a. infusions of norepinephrine (0.5, 1 or 4 micrograms base/min). In the cutaneous vasculature, DA produced large artery, small vessel and large vein constrictions. The small vessels and large veins constricted proportionately more than the large arteries. The pattern of constriction produced by DA along the cutaneous vascular tree was similar to that produced by norepinephrine. The forelimb vasoconstriction produced by both DA and norepinephrine was abolished completely by treatment with phentolamine, indicating that both agents produce vasoconstriction mediated by stimulation of alpha adrenoceptors. The failure of DA to produce vasodilation after phentolamine suggests that there are no vascular or DA1-subtype DA receptors in the canine forelimb vasculature. The local i.a. infusion of SKF 82526, a selective DA1-receptor agonist, for 3 min produced cutaneous and skeletal muscle dilation which could be antagonized by either sulpiride or phentolamine. This neurogenic dopaminergic vasodilation produced by SKF 82526 (25, 50 or 100 micrograms base/min) was converted into vasoconstriction after ganglionic blockade. Inasmuch as SKF 82526 does not activate presynaptic or DA2-subtype DA receptors, the neurogenic vasodilation caused by SKF 82526 may be due to a dopaminergic inhibition of ganglionic transmission. Whereas the results of our study fail to provide any evidence for the presence of vascular DA (DA1) receptors in the canine forelimb, they show that SKF 82526, a DA1-receptor agonist, produces neurogenic vasodilation probably by activating ganglionic DA receptors.     

Defective dopamine-1 receptor adenylate cyclase coupling in the proximal convoluted tubule from the spontaneously hypertensive rat.

The natriuretic effect of DA-1 agonists is less in the spontaneously hypertensive rat (SHR) than its normotensive control, the Wistar-Kyoto rat (WKY). To determine a mechanism of the decreased effect of DA-1 agonists on sodium transport, DA-1 receptors in renal proximal convoluted tubule (PCT) were studied by radioligand binding and by adenylate cyclase (AC) determinations. Specific binding of 125I-SCH 23982 (defined by 10 microM SCH 23390, a DA-1 antagonist) was concentration dependent, saturable, and stereoselective. The dissociation constant, maximum receptor density, and DA-1 antagonist inhibition constant were similar in SHR and WKY. The apparent molecular weight of the DA-1 receptor determined by the photoaffinity D1 probe 125I-MAB was also similar in WKY and SHR. However, DA-1 agonists competed more effectively for specific 125I-SCH 23982 binding sites in WKY than in SHR. Basal as well as forskolin, parathyroid hormone, GTP and Gpp(NH)p-stimulated-AC activities were similar. In contrast DA-1 agonists (fenoldopam, SKF 38393, SND 911C12) stimulated AC activity to a lesser extent in SHR. GTP and Gpp(NH)p enhanced the ability of DA-1 agonists to stimulate AC activity in WKY but not in SHR. These data suggest a defect in the DA-1 receptor-second messenger coupling mechanism in the PCT of the SHR.     

Muscarinic stimulation of adenylate cyclase activity of rat olfactory bulb. I. Analysis of agonist sensitivity

In rat olfactory bulb, stimulation of muscarinic receptors activates adenylate cyclase. In the present study we have examined a variety of muscarinic receptor stimulants to characterize the agonist profile of this response. Analysis of agonist concentration-response curves revealed the following rank order of potency: oxotremorine-M greater than oxotremorine greater than BM5 greater than acetylcholine greater than carbachol = methacholine greater than (+/-)muscarine greater than arecoline greater than pilocarpine greater than RS 86 greater than McN-A-343 greater than bethanechol. Acetylcholine, oxotremorine-M, carbachol, (+/-)muscarine and metacholine behaved as full agonists, whereas the other stimulants displayed lower efficacies. The slope values of the concentration-response curves were close or equal to 1, except those of the carbachol and pilocarpine curves, which showed values significantly lower than 1. Moreover, the slope of the pilocarpine curve was differentially changed by the M1 antagonist pirenzepine and the M2 antagonist AF-DX 116. The agonist profile of the muscarinic stimulation of adenylate cyclase in the olfactory bulb correlated well with that exhibited by the muscarinic inhibition of the enzyme activity in the striatum, suggesting that the two responses are mediated by a similar receptor subtype. Sodium ion modulated the agonist affinity for both adenylate cyclase-coupled receptor systems.     

Utilization of spectral absorption for measurement of adenylate cyclase activity

The purpose of this study was to improve our previously described enzymatic fluorometric assay of the adenylate cyclase activity. Using physicochemical characteristics of NADPH, of which a 0.1 mmol/L solution would have an optical density of 0.627, we measured the adenylate cyclase activity by the spectral absorption of NADPH. The assay consists of two parts: pharmacological modulation of adenylate cyclase and measurement of newly synthesized cyclic AMP. The latter part involves four steps: enzymatic destruction of noncyclic adenine nucleotides and phosphorylated metabolites, conversion of cyclic AMP to ATP, amplification of ATP by enzymatic cycling, and measurement of NADPH with spectral absorption, which is generated in proportion to initial cyclic AMP levels. This new assay was tested in membrane preparations made from rat hearts in comparison with the previously described fluorometric assay. We obtained identical results by spectrophotometry and fluorometry with high reproducibility. Because the fluorometric assay possesses a high sensitivity while the spectrophotometric method is advantageous because of its wide analytical range of cyclic AMP measurement, combination of fluorometric and spectrophotometric methods may offer a convenient way to measure the adenylate cyclase activities in various samples.     

Effect of age and posture on human lymphocyte adenylate cyclase activity

1. A number of age-related changes have been reported in the catecholamine-adrenoceptor-adenylate cyclase system. Most of the data available on these alterations come from resting subjects; the response to acute stress may provide additional insights into the age effect on these responses. 2. We measured supine and 10 min upright plasma noradrenaline and lymphocyte adenylate cyclase activity in ten healthy elderly subjects (age 66-80 years) and seven healthy young subjects (age 27-34 years). 3. Isoprenaline stimulation of lymphocyte adenylate cyclase activity was not significantly different between supine and upright positions or between elderly and young subjects. There was a marked increase in forskolin-stimulated adenylate cyclase activity in the upright posture in both elderly and young subjects. The increment over supine levels was 70% in the elderly (P less than 0.025) and 73% in the young (P less than 0.05). This enhanced forskolin activity was not seen in two young subjects who became syncopal. 4. These data suggest that enhanced forskolin-stimulated adenylate cyclase activity occurs after 10 min of upright posture in both elderly and young subjects, and may be relevant to immediate blood pressure regulation. We were unable to demonstrate any age-related differences in these acute adrenergic responses.     

Castration blocks chronic sulpiride-induced desensitization of striatal D1 receptor-stimulated adenylate cyclase activity in male rats

Gonadal hormones have been shown to modulate adaptive responses of the mesostriatal dopaminergic system to antipsychotic challenge. We examined the role of endogenous gonadal steroids in the regulation of D1 receptor function after chronic treatment with sulpiride, a D2 specific antagonist. Chronic sulpiride treatment induced a desensitization of striatal D1 receptor-simulated adenylate cyclase activity in intact male rats with no change in the number of D1 or D2 receptors. This desensitization of D1-stimulated adenylate cyclase activity was expressed as a decrease in Vmax with no change in the activation constant. Castration of male rats blocked the chronic sulpiride-induced desensitization of D1 receptor function. Castration of male rats also resulted in a decrease in the number of D1 receptors as measured by [3H]SCH23390 binding. Ovariectomy of female rats had no effect on striatal D1 receptor-stimulated adenylate cyclase activity. Preliminary studies showed no effect of chronic sulpiride treatment on D1 receptor function in intact or ovariectomized female rats. We conclude that testicular hormones have a permissive effect on the expression of the chronic sulpiride-induced desensitization of D1 receptor function.     

Human gastric mucosal adenylate cyclase activity: effects of various cytoprotective prostaglandins

Several prostaglandins prevent ulcer formation (called cytoprotection) by a mechanism other than inhibition of gastric acid secretion. One suggestion is that they increase cyclic AMP in non-parietal cells. A variety of prostaglandins with potent cytoprotective properties were tested for their capacity to modulate adenylate cyclase activity in homogenates of human gastric mucosa. Prostaglandin E2, prostacyclin (PGI2) and 15(S)-methyl-PGE2 stimulated the cyclase in human gastric mucosal biopsy specimens in a dose-dependent manner. Cytoprotective prostaglandins without antisecretory properties such as PGF2 beta were also able to activate the enzyme system dose-dependently. In contrast, cytoprotective prostaglandins such as PGD2, the PGE1-analogue, SC-29333, and the prostaglandin-like compound C83 did not stimulate human gastric adenylate cyclase. Whereas PGD2 did not modulate enzyme activity at all, SC-29333 and C83, at concentrations greater than 10 mumol/l, inhibited basal and PGE2-stimulated enzyme activities. These studies suggest that cyclic AMP is not directly related to the cytoprotective effect of prostaglandins, at least in human gastric mucosa.     

The effects of PGE2 on vasopressin--and guanine nucleotide-mediated adenylate cyclase activity in toad bladder membrane

PGE2 inhibited 10 m U/ml vasopressin-induced osmotic water flow of the toad bladder at 2 X 10(-8) M. PGE2 suppressed vasopressin-mediated cyclic AMP accumulation in epithelial cells and also vasopressin-mediated adenylate cyclase activity in a crude homogenate of the cells. However, PGE2 had no effect on cyclic AMP dependent and independent protein phosphorylation. These findings indicate that PGE2 inhibits vasopressin-induced water flow mainly through suppression of adenylate cyclase activity, and that the role of PGE2 at that point in the reaction leading to increased water permeability following cyclic AMP production may be slight. Under conditions in which the hormone and substrate are depleted, PGE2 and guanine nucleotides, such as GTP and Gpp(NH)p, additively bring about an increase in adenylate cyclase activity.     

ATP receptor regulation of adenylate cyclase and protein kinase C activity in cultured renal LLC-PK1 cells.

In cultured intact LLC-PK1 renal epithelial cells, a nonhydrolyzable ATP analogue, ATP gamma S, inhibits AVP-stimulated cAMP formation. In LLC-PK1 membranes, several ATP analogues inhibit basal, GTP-, forskolin-, and AVP-stimulated adenylate cyclase activity in a dose-dependent manner. The rank order potency of inhibition by ATP analogues suggests that a P2y type of ATP receptor is involved in this inhibition. The compound ATP gamma S inhibits agonist-stimulated adenylate cyclase activity in solubilized and in isobutylmethylxanthine (IBMX) and quinacrine pretreated membranes, suggesting that ATP gamma S inhibition occurs independent of AVP and A1 adenosine receptors and of phospholipase A2 activity. The ATP gamma S inhibition of AVP-stimulated adenylate cyclase activity is not affected by pertussis toxin but is attenuated by GDP beta S, suggesting a possible role for a pertussis toxin insensitive G protein in the inhibition. Exposure of intact LLC-PK cells to ATP gamma S results in a significant increase in protein kinase C activity. However, neither of two protein kinase C inhibitors (staurosporine and H-7) prevents ATP gamma S inhibition of AVP-stimulated adenylate cyclase activity, suggesting that this inhibition occurs by a protein kinase C independent mechanism. These findings suggest the presence of functional P2y purinoceptors coupled to two signal transduction pathways in cultured renal epithelial cells. The effect of P2y purinoceptors to inhibit AVP-stimulated adenylate cyclase activity may be mediated, at least in part, by a pertussis toxin insensitive G protein.     

Desensitization of adenylate cyclase and down regulation of beta adrenergic receptors after in vivo administration of beta agonist

In vitro incubation of cells with catecholamines leads to both down regulation of beta adrenergic receptor number and desensitization of agonist-stimulated adenylate cyclase activity. These same parameters, down regulation of beta adrenergic receptor number and desensitization of adenylate cyclase activity were assessed in rat lung membranes after in vivo administration of metaproterenol, a beta-2 selective agonist. In vivo treatment with metaproterenol leads to: 1) reduced beta adrenergic receptor number; 2) reduced isoproterenol-stimulated adenylate cyclase activity; 3) unaffected NaF or 5'-guanylylimidodiphosphate-stimulated adenylate cyclase activity; and 4) reduced affinity of the receptor for isoproterenol similar to the affinity observed in the presence of 5'-guanylylimidodiphosphate. The date suggest that in vivo metaproterenol administration results in an uncoupled receptor-adenylate cyclase complex. The effects of in vivo administration of the glucocorticoid, methylprednisolone, to metaproterenol-pretreated animals were also assessed. Glucocorticoid treatment was associated with 1) increased beta adrenergic receptor number in rats in which the receptors have been down regulated, 2) increased isoproterenol responsiveness in agonist-desensitized rats and 3) no effect on agonist affinity in desensitized animals. These data suggest that the restoration of agonist responsiveness by glucocorticoids in the catecholamine refractive state is not simply a reversal of receptor down regulation or adenylate cyclase desensitization.     

Effect of dobutamine on adenylate cyclase activity in rabbit renal pelvis and ureter

The adenylate cyclase activity in the rabbit renal pelvis and ureter was measured by the method of Salomon et al. (1974). The dobutamine-induced increase of adenylate cyclase activity is more prominent in the renal pelvis including the pacemaker region than in the ureter that is non-pacemaker region in the upper urinary tract. Therefore, it is thought that the pacemaker region is different from other regions in the upper urinary tract with regard to responses to dobutamine.     

Decreased bioactivity of the guanine nucleotide-binding protein that stimulates adenylate cyclase in hearts from cardiomyopathic Syrian hamsters.

We investigated regulation of cardiac adenylate cyclase in 29-d-old BIO 14.6 Syrian hamsters, which inherit cardiomyopathy as an autosomal recessive trait. Pharmacologic stimulation of adenylate cyclase in cardiac membranes with isoproterenol, fluoride ion, guanine nucleotide, forskolin, and manganous ion indicated that there was defective coupling of the guanine nucleotide-binding protein that stimulates adenylate cyclase (Gs) to adenylate cyclase. Cyc complementation assays revealed congruent to 50% less Gs activity in cardiac and skeletal muscle from cardiomyopathic hamsters. Despite this decrease in functional Gs, there were no changes in immunologic levels of the alpha-subunit of Gs (alpha Gs) or in levels of mRNA encoding alpha Gs. The defect in Gs bioactivity was limited to cardiac and skeletal muscle, occurred only in animals homozygous for the dystrophic trait, and was demonstrable before any cardiac abnormalities were evident on light microscopy. By contrast, cardiac levels of beta-adrenergic receptors were not different in cardiac membranes from BIO 14.6 hamsters. We conclude that a functional defect in alpha Gs may contribute to a contractile abnormalities in the cardiomyopathic BIO 14.6 hamster. However, the etiology of the alpha Gs defect remains obscure.     

Adenosine A1 receptors inhibit adenylate cyclase activity and neurotransmitter release and hyperpolarize pyramidal neurons in rat hippocampus

Adenosine is a neuromodulator with multiple actions upon the physiology and biochemistry of the brain. Although the receptors that inhibit synaptic transmission and regulate cyclic AMP formation have been well characterized in terms of their pharmacological properties, the receptor(s) that mediate the postsynaptic actions of adenosine have not. We have used the adenosine-mediated inhibition of low calcium bursting in the rat hippocampal slice preparation as a measure of a postsynaptic effect of adenosine. Low calcium bursting consists of repetitive spiking evoked by antidromic stimulation of the CA1 pyramidal neurons and can be suppressed by adenosine. In the present study, we compared the effects of a selective adenosine A1 receptor antagonist, 8-cyclopentyltheophylline, on functional responses to adenosine in hippocampal slices. Its apparent affinities for the receptors mediating the following effects were increases in cyclic AMP formation (1700 nM), decreases in cyclic AMP formation after forskolin pretreatment (57 nM), inhibition of excitatory synaptic responses (42 nM), inhibition of repetitive spiking (45 nM), and it was a competitive antagonist for all responses tested. These data demonstrate that inhibition of transmitter release, adenylate cyclase and low calcium bursting in the hippocampus are all mediated via adenosine receptors that have pharmacological properties similar to the A1 receptor and that 8-cyclopentyltheophylline has a high degree of selectivity for this receptor as opposed to the A2 receptor that mediates increases in cyclic AMP formation.     

Gallamine allosterically antagonizes muscarinic receptor-mediated inhibition of adenylate cyclase activity in the rat myocardium

The ability of gallamine to modify muscarinic receptor binding properties and to antagonize muscarinic receptor-mediated inhibition of adenylate cyclase activity was investigated in the rat myocardium. Gallamine caused parallel shifts to the right in the dose-response curves for inhibition of adenylate cyclase activity by the highly efficacious muscarinic agonist oxotremorine-M and the partial agonist Bm 5 [N-methyl-N-(1-methyl-4-pyrrolidino)-2-butynyl acetamide]. The nature of this effect was inconsistent with competitive inhibition, but could be explained by allosteric antagonism. Similar dissociation constants of 0.52 and 0.83 microM were estimated for gallamine on the basis of its ability to antagonize responses to oxotremorine-M and Bm 5, respectively. The maximum shift in the dose-response curve of Bm 5 caused by gallamine was 90-fold, whereas that of oxotremorine-M was only 49-fold. When measured by inhibition of the binding of the specific muscarinic antagonist [3H]N-methylscopolamine, the dissociation constant of gallamine was estimated to be 1.1 microM. The present results illustrate good agreement between the ability of gallamine to modify muscarinic receptor binding properties and to antagonize muscarinic receptor-mediated inhibition of adenylate cyclase activity in the rat heart.     

Pharmacological characterization of muscarinic receptors mediating inhibition of adenylate cyclase activity in the rat retina

Incubation of the rat retina with acetylcholine resulted in about a 20 to 30% decrease of basal cyclic AMP accumulation. Oxotremorine, arecoline, [4-hydroxy-2-butynyl]trimethylammonium chloride, m-chlorocarbanilate and carbachol also inhibited cyclic AMP accumulation. Nicotine had no effect. The response was blocked by atropine and pirenzepine but not gallamine. Intraocular injection of pertussis toxin 72 hr before testing also blocked the response to acetylcholine. The presence of forskolin exaggerated the response to acetylcholine. Intraocular injection of the cholinotoxin AF64A resulted in apparent supersensitivity of the response to acetylcholine. Our results suggest that rat retina contains muscarinic M1 receptors that are coupled negatively to adenylate cyclase.     

Effect of chilling on platelet cyclic adenosine 3:5-monophosphate and adenylate cyclase activity

Exposure of platelets to 1 C led to a transient increase in cyclic AMP levels (determined either by a protein binding method or by radioimmunoassay) within five to ten minutes reaching a maximum 10 to 15 minutes after chilling was begun and returning subsequently to baseline values. Addition of EDTA to the platelet suspension medium prevented this increase. Rewarming at 37 C produced a sudden reduction in platelet cyclic AMP. To determine whether the cold-induced increase in cyclic AMP was due to a transient stimulation of platelet adenylate cyclase or a rapid inhibition of phosphodiesterase, these enzymes were assayed in ruptured platelet suspensions. Platelet adenylate cyclase activity was found to possess certain characteristics similar to those of the enzyme derived from other sources but there was a marked potentiation of fluoride-stimulated adenylate cyclase activity by 0.001 M EDTA. This effect was limited to low EDTA concentrations. Exposure of platelets to 1 C for up to 60 minutes did not increase adenylate cyclase activity but lowered it substantially compared with controls kept at room temperature. Phosphodiesterase activity at 1 C was depressed sooner and to a greater extent than was adenylate cyclase. The transient rise in cyclic AMP levels in chilled platelets appears to be due to a disproportionate reduction of cyclic nucleotide phosphodiesterase activity.     

Intrarenally produced angiotensin II opposes the natriuretic action of the dopamine-1 receptor agonist fenoldopam in rats.

There are reports of an increase in renin release after the administration of fenoldopam which probably results from the activation of dopamine (DA)-1 receptors located on juxtaglomerular cells in the kidney. However, the functional significance of this finding in terms of modulating the tubular response to DA-1 receptor stimulation remains to be determined. In this study we have examined the effect of an increase in renin-angiotensin system activity during the administration of fenoldopam on the natriuretic and diuretic action of this compound. Intravenous infusion of fenoldopam (0.5 microgram/kg/min) for 30 min produced significant increases in urine output and urinary sodium excretion without causing any changes in glomerular filtration rate, renal blood flow and mean arterial blood pressure, a phenomenon suggestive of a direct tubular site of action. In animals treated with the angiotensin converting enzyme inhibitor captopril, both the diuretic and natriuretic effects of fenoldopam were potentiated markedly. In comparison with fenoldopam infusion in control animals, urine output, urinary sodium excretion and potassium excretion increased by approximately 4-fold (375 +/- 24 vs. 97 +/- 3 microliters/30 min, P less than .01), 9-fold (50 +/- 5 vs. 6 +/- 1 microEq/30 min, P less than .001) and 2-fold (46 +/- 8 vs. 24 +/- 2 microEq/30 min, P less than .05), respectively, in animals receiving a bolus injection of captopril (1 mg/kg i.v.) 30 min before onset of fenoldopam infusion. Whereas no significant changes in renal blood flow occurred when fenoldopam was given to control rats, in animals treated with captopril, fenoldopam produced a modest but significant increase in renal blood flow.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of adenylate cyclase in the locus coeruleus mediates the hypnotic response to an alpha 2 agonist in the rat.

Recently, we determined that the transduction mechanism for the hypnotic response to dexmedetomidine, a highly selective alpha 2 agonist, resides in the locus coeruleus (LC) of the rat. Candidates for the effector mechanism of this alpha 2 adrenoceptor-mediated hypnotic response include inhibition of adenylate cyclase, which has been shown to be pivotal to the cellular response of alpha 2 agonists in some, but not in all, cases. The LC of rats were stereotaxically cannulated with an indwelling catheter, and after the 2nd day, the hypnotic response to 7 micrograms of dexmedetomidine into the LC (an effective hypnotic dose for 95% of animals) was tested. Other groups of rats were pretreated with the permeable nonhydrolyzable cyclic AMP (cAMP) analog, dibutyryl cAMP (dB cAMP), at a dose of 0.2 to 1.2 ng into the LC, or 2.75 to 275 micrograms.kg-1 i.p. rolipram, a cAMP-specific phosphodiesterase inhibitor, and the hypnotic response to 7 micrograms of dexmedetomidine into the LC was tested. Both dB cAMP and rolipram reversed the hypnotic response to dexmedetomidine. To test for the specificity of these hypnotic-reversing perturbations, rats were pretreated with Rp-adenosine-3',5'-cyclic phosphorothioate, a cAMP-dependent protein kinase inhibitor, and the experiments were repeated. The hypnotic-reversing property of either dB cAMP or rolipram could be prevented by blocking cAMP-dependent protein kinase ("A" kinase) activity with Rp-adenosine-3',5'-cyclic phosphorothioate.(ABSTRACT TRUNCATED AT 250 WORDS)