Comparative effects of cytochalasin D on the protein discharge induced by alpha- and beta-adrenergic or cholinergic agonists in rat exorbital lacrimal glands

Cytochalasin D altered the kinetics of peroxidase and radiolabeled protein discharge from rat exorbital lacrimal glands in vitro, in response to various secretagogues. The changes were different with each inducer. The discharge due to isoproterenol was immediately inhibited by 95%; the discharge evoked by noradrenaline via alpha-adrenergic receptors was progressively reduced and was inhibited by 50% after 30 min, whereas that evoked by carbachol was not influenced during the initial discharge period and was diminished by only 30% after 30 min. When calcium was removed from the incubation medium, the secretory responses were lowered and the inhibitory effect of cytochalasin D was still observed. The rate of protein discharge inhibition was related to the dose and was maximal with 2 X 10(-6) M cytochalasin D when the discharge resulted from cholinergic, alpha- or beta-adrenergic or dibutyryl cAMP stimulation. Cytochalasin D did not impair cellular energetics nor other stimulations induced through muscarinic or adrenergic receptors. Cytochalasin D effects could be related to interaction with actin, leading to the inhibition of the release of proteins into the incubation medium following the activation of the adrenergic system.     

Beta- and alpha-adrenergic receptors are involved in regulating type II thyroxine 5'-deiodinase activity in the rat Harderian gland.

The role of alpha- and beta-adrenergic receptors in regulation of rat Harderian gland type II thyroxine 5'-deiodinase (5'-D) activity was investigated. Our results show that isoproterenol, a beta-adrenergic agonist, and phenylephrine, an alpha-adrenergic agonist, elicited increases in Harderian gland 5'-D activity. The activation was dependent on the time and the dose of the drug. Other adrenergic agonists, i.e., norepinephrine, methoxamine or terbutaline, also clearly increased the enzyme activity. Moreover, administration of propranolol, a beta-adrenergic blocker, or prazosin, an alpha-adrenergic blocker, completely prevented the activation of the enzyme induced by norepinephrine. Results show a clear regulation by adrenergic mechanisms of 5'-D activity in the rat Harderian gland, where alpha- and beta-adrenergic receptors appear to be involved.     

Alpha-2 adrenergic agonists inhibit basal and stimulated osmotic water permeability in toad skin

The effect of selective alpha adrenergic agonists and antagonists on osmotic water permeability (Posm) across isolated skins of Bufo arenarum toads was investigated. Clonidine, an alpha-2 agonist, inhibited basal Posm and oxytocin, isoproterenol and theophylline stimulated Posm, but did not alter the hydrosmotic effect of exogenous cAMP. Blockade of the effect of clonidine on basal and stimulated Posm by the selective alpha-2 antagonist yohimbine supports the hypothesis that the inhibitory effect is mediated by the stimulation of alpha-2 adrenergic receptors.     

An Examination of the Involvement of Phospholipases A2 and C in the α-Adrenergic and γ-Aminobutyric Acid Receptor Modulation of Cyclic AMP Accumulation in Rat Brain Slices

Experiments were undertaken to define the role of two calcium-associated enzyme systems in modulating transmitter-stimulated production of cyclic nucleotides in rat brain. Cyclic AMP (cAMP) accumulation was examined in cerebral cortical slices using a prelabeling technique. The enhancement of isoproterenol-stimulated cAMP production by alpha-adrenergic and gamma-aminobutyric acid-B (GABAB) agonists was reduced by exposing the tissue to EGTA, a chelator of divalent cations, or quinacrine, a nonselective inhibitor of phospholipase A2. Likewise, chronic (2 weeks) administration of corticosterone decreased the alpha-adrenergic and GABAB receptor modulation of second messenger production. Neither cyclooxygenase nor lipoxygenase inhibitors selectively influenced the facilitating response of alpha-adrenergic and GABAB agonists. Other experiments revealed that although norepinephrine and 6-fluoronorepinephrine stimulated inositol phosphate (IP) production in cerebral cortical slices with potencies equal to those displayed in the cyclic nucleotide assay, selective alpha 1-adrenergic agonists were less efficacious on IP formation and were without effect in the cAMP assay. Conversely, a selective alpha 2-adrenergic receptor agonist facilitated the cAMP response to a beta-adrenergic agonist without affecting IP formation. The rank orders of potency of a series of alpha-adrenergic antagonists suggest that IP accumulation is mediated solely by alpha 1-adrenergic receptors, whereas the augmentation of cAMP accumulation is regulated by a mixed population of alpha-adrenergic sites. The results suggest that the alpha-adrenergic and GABAB receptor-mediated enhancement of isoproterenol-stimulated cAMP formation appears to be more closely associated with phospholipase A2 than phospholipase C and may be mediated by arachidonate or some other fatty acid.     

Absence of alpha-2 adrenergic effects on cAMP production in a genital tract smooth muscle cell line

This study sought to evaluate alpha-2 and beta adrenergic modulation of cAMP production in the DDT1 MF-2 transformed smooth muscle myocyte. After stimulation with forskolin or adrenergic agonists with or without subtype specific antagonists, cAMP production was determined. These experiments confirmed an increase of cAMP in response to forskolin, isoproterenol, epinephrine, and norepinephrine; the adrenergic stimulation was inhibited by propranolol. On the other hand, the alpha-2 agonist clonidine did not inhibit cAMP production. Likewise, alpha-2 receptor blockade did not increase cAMP production in response to epinephrine. These studies, therefore, suggest that the DDT1 MF-2 myocyte does not contain a significant population of functional alpha-2 adrenergic receptors.     

Additive competitive interaction of verapamil and quinidine at alpha-adrenergic receptors of isolated cardiac guinea pig myocytes and human platelets

Recent clinical work has questioned the safety of a combined therapy of oral quinidine and intravenous verapamil, because some patients were reported to react with severe hypotension probably due to drug interactions with vascular alpha-adrenergic receptors. In order to obtain further quantitative information on the underlying mechanism, we used the radioligands (3H)-prazosin and (3H)-yohimbine to perform binding studies on intact cells, with predominantly alpha-1 (isolated myocytes) or alpha-2 subtypes (human platelets) of adrenergic receptors. Our studies confirm that both verapamil and quinidine possess a distinct alpha-adrenergic receptor blocking activity and do not discriminate between the alpha-1 and alpha-2 subtype (Ki-values were between 0.24-0.28 mumol/l for alpha-1 receptors and 0.49-0.50 mumol/l for alpha-2 receptors). Their interaction was competitive and in the presence of both drugs inhibition of radioligand binding was additive. The alpha-adrenergic blockade by verapamil was stereospecific as D-verapamil increased the dissociation constant of the radioligand to a much lesser degree than L-verapamil (Ki = 1.67 +/- 0.29 mumol/l for D-verapamil). The calcium channel blocker nitrendipine, a 1,4-dihydropyridine derivative, did not show any competition up to concentrations of 10 mumol/l. Our results thus give evidence that verapamil and quinidine have already at therapeutic blood levels significant alpha-adrenergic blocking activities which may be of clinical interest. In addition our results show that adult cardiac myocytes are very well suited for pharmacological adrenergic interaction studies.     

Development and properties of the secretory response in rat sweat glands: relationship to the induction of cholinergic function in sweat gland innervation

Previous studies suggest that the sympathetic innervation of the sweat glands in the rat is initially noradrenergic and during development undergoes a transition in neurotransmitter phenotype to become cholinergic. To characterize this system and its development further, we have examined the adrenergic and cholinergic components of the secretory response in adult and immature rats and have studied the onset of sweating in the plantar sweat glands of developing rats. Stimulation of the sciatic nerve in adult rats elicited a secretory response which was completely blocked by the cholinergic antagonist, atropine, and was unaffected by adrenergic antagonists, indicating that nerve-evoked secretion was cholinergic. In adult rats, the sweat glands were quite sensitive to cholinergic agonists. In addition to acetylcholine, the mature sweat gland innervation contains vasoactive intestinal peptide (VIP). In some rats, the injection of VIP alone elicited a secretory response which was blocked by atropine, suggesting that the response to VIP was mediated cholinergically. In contrast to cholinergic agonists, the glands responded relatively infrequently and with reduced volumes of sweat to the alpha- and beta-adrenergic agonists 6-fluoronorepinephrine and isoproterenol. However, when VIP, which is a potent vasodilator, was simultaneously injected with adrenergic agonists, glands in many of the injected footpads exhibited a secretory response. The response to adrenergic agonists in combination with VIP was reduced by atropine and by phentolamine plus propranolol, but was blocked completely only by a combination of the three antagonists, indicating that both adrenergic and cholinergic mechanisms were involved. In immature rats, sweating evoked by nerve stimulation first appeared at 14 days of age in 25% of the rats tested. Both the percentage of rats sweating and the number of active glands increased rapidly. At 16 days, 50% of the rats tested exhibited some active glands, and by 21 days all rats tested exhibited a secretory response. In 16-day-old rats, nerve-evoked sweating was almost completely inhibited by local injection of 1 microM atropine, but was unaffected by phentolamine and propranolol in concentrations up to 10 microM. Similarly, the glands were sensitive to 10 microM muscarine, but they exhibited no secretory response to the alpha-adrenergic agonists, clonidine and 6-fluoronorepinephrine, nor to the beta-adrenergic agonist, isoproterenol, at concentrations up to 50 microM. The simultaneous injection of VIP with adrenergic agonists did not reveal an adrenergically mediated secretory response in 16-day-old animals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Multiple second messenger pathways of alpha-adrenergic receptor subtypes expressed in eukaryotic cells

The alpha-adrenergic receptors mediate the effects of epinephrine and norepinephrine on cellular signaling systems via guanine nucleotide binding regulatory proteins (G-proteins). Three alpha-adrenergic receptor subtypes have been cloned: the alpha 1, the alpha 2-C10, and the alpha 2-C4 adrenergic receptors. To investigate functional differences between the different subtypes, we assessed the ability of each to interact with adenylyl cyclase and polyphosphoinositide metabolism by permanently and transiently expressing the DNAs encoding the alpha 1, the alpha 2-C10, and the alpha 2-C4 adrenergic receptors in cells lacking endogenous alpha-adrenergic receptors. Both alpha 2-C10 and alpha 2-C4 couple primarily to inhibition of adenylyl cyclase and to a lesser extent to stimulation of polyphosphoinositide hydrolysis. alpha 2-C10 inhibits adenylyl cyclase more efficiently than alpha 2-C4. Effects of the alpha 2-adrenergic receptors on adenylyl cyclase inhibition and on polyphosphoinositide hydrolysis are both mediated by pertussis toxin-sensitive G-proteins. The major coupling system of the alpha 1-adrenergic receptor is activation of phospholipase C via a pertussis toxin-insensitive G-protein. alpha 1-Adrenergic receptor stimulation can also increase intracellular cAMP by a mechanism that does not involve direct activation of adenylyl cyclase. As with the muscarinic cholinergic receptor family our results show that each of the alpha-adrenergic receptor subtypes can couple to multiple signal transduction pathways and suggest several generalities about the effector coupling mechanisms of G-protein-coupled receptors.     

The role of beta receptors in the regulation of renin release.

The effect of the primarily beta 2 type adrenergic receptor stimulating terbutaline (10(-7)--10(-5) M) and of the beta 1 and beta 2 type adrenergic receptor stimulating isoproterenol (10(-7)--10(-5) M) was studied on renin release from incubated slices of renal cortex. Renin release and cAMP content of the slices were significantly higher in the presence of both terbutaline and isoproterenol. A logarithmic dose--response relationship was shown to be present between the beta mimetics and the renin concentration in the medium, and the cAMP content of tissue slices. In equal doses isoproterenol was about 1.5 times more potent than terbutaline. No change was seen in the renin content of the tissue slices. The results supports the view that beside the beta 1 type adrenergic receptors of the renal cortex--even if to a lesser extent--the beta 2 type adrenergic receptors, too, are involved in the regulation of renin release.     

Alpha adrenergic stimulation reduces cyclic adenosine 3',5'-monophosphate generation in rabbit myometrium by two mechanisms

Rabbit myometrium contains postsynaptic alpha-1, alpha-2, and beta-2 adrenoreceptors. The response to endogenous catecholamines depends on the summation of interactions at these receptors and is influenced by the hormonal environment. Estrogen treatment of ovariectomized rabbits increases the alpha adrenergic contractile response whereas progesterone treatment of estrogen primed animals results in a predominance of the beta adrenergic response, which is inhibition of contractions. Of the receptor subtypes, only the alpha-2 receptor concentration is increased at physiological estrogen concentrations. However, alpha-2 receptors have not been shown to be directly involved in myometrial contraction, which appears to be mediated solely by alpha-1 adrenergic interactions. To test whether alpha-2 receptors might indirectly affect contraction by opposing interactions at the beta receptor, we examined the ability of alpha adrenergic stimulation to reduce myometrial cyclic adenosine 3',5'-monophosphate (cAMP) generation. We find that alpha-2 receptors inhibit myometrial ade adenylate cyclase through the guanine nucleotide regulatory protein, Gi. In addition, we find that activation of alpha-1 receptors also reduces cAMP generation. This interaction, which can be demonstrated in the absence but not the presence of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, does not appear to be mediated through Gi. These findings illustrate the complexity of adrenergic interactions in tissues containing several adrenergic subtypes.     

Regulation of platelet-activating factor receptor and platelet-activating factor receptor-mediated biological responses by cAMP in rat Kupffer cells

Treatment of cultured Kupffer cells with the beta-adrenergic agonist isoproterenol (10 microM) for a short period of time (30 min) attenuated the subsequent platelet-activating factor (PAF)-induced arachidonic acid release and cyclooxygenase-derived eicosanoid (e.g. thromboxane B2 and prostaglandin E2) production. This effect of isoproterenol was highly specific since the alpha-adrenergic agonist phenylephrine and the beta-adrenergic antagonist propranolol had no effect on the stimulatory effect of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC). The inhibitory effect of isoproterenol on the AGEPC-induced arachidonic acid release was demonstrated through the use of a specific beta-adrenergic subtype agonist and antagonist to be mediated by beta 2-adrenergic receptors on Kupffer cells. These inhibitory effects of isoproterenol can be mimicked by dibutyryl cAMP but not by dibutyryl cGMP, suggesting that a cAMP-dependent mechanism is likely involved in the regulatory action of isoproterenol. Ligand binding studies indicated that short term (i.e. 30 min) treatment of the cultured Kupffer cells with either isoproterenol or dibutyryl cAMP had no effect on the specific [3H]PAF binding. However, long term incubation (9-24 h) with dibutyryl cAMP caused down-regulation of the PAF receptors in rat Kupffer cells. Forskolin (0.1 mM), an adenylyl cyclase activator, down-regulated the surface expression of the AGEPC receptors more rapidly, decreasing the specific [3H]AGEPC binding by approximately 40% within 2 h. The receptor regulatory effect of dibutyryl cAMP and forskolin was time- and concentration-dependent. These observations suggest that a cAMP-dependent mechanism coupled with beta 2-adrenergic receptors may have important regulatory effects on the PAF receptor and post-receptor signal transducing mechanisms for PAF in hepatic Kupffer cells.     

The hepatic adrenergic receptors

Summary The presence of both alpha- and beta-adrenergic receptors in liver designated the hepatic plasma membrane as a useful tool for the elucidation of the mechanisms by which the hormonal signal is transfered through the membrane via a coupling system to an amplifying entity. This process is well documented for the beta-adrenergic receptor which is linked to adenylate cyclase, whereby it modulates the cyclic AMP level. Much less is known about the alpha-adrenergic receptor.Recently, two factors have contributed to a renewed interest in alpha- and beta-adrenergic receptors in liver: i) The fact that activation of glycogenolysis in isolated liver parenchymall cells by epinephrine may be mediated by either alpha- or beta-adrenergic receptors, depending on the species or on the state of nutrition, and not only by beta-adrenergic receptors as previously thought. ii) The existence of specific adrenergic agonists and antagonists radiolabeled to a high specific activity which has permitted the characterization of adrenergic receptors in terms of nature, number, affinity and regulation.The present review will be devoted to the recent progress made in the physiological, pharmacological and biochemical characterization of alpha- and beta-adrenergic receptors in the liver.     

Activation of alpha-1 adrenergic receptors modulates the control of left/right sidedness in rat embryos.

Presomite stage rat embryos were cultured for 45-49 hr with medium containing various adrenergic agonists and antagonists. L-Norepinephrine but not D-norepinephrine (several orders of magnitude less potent than the L-isomer at alpha-1 adrenergic receptors) resulted in a dose-dependent increase of situs inversus similar to that found for phenylephrine, an alpha-1 adrenergic agonist. Prazosin, an alpha-1 adrenergic antagonist, inhibited phenylephrine-induced situs inversus in a dose-dependent manner. Neither dexmedetomidine, an alpha-2 adrenergic agonist, nor isoproterenol, a beta adrenergic agonist, caused situs inversus. These results provide pharmacological evidence that stimulation of alpha-1 but not of alpha-2 and beta adrenergic receptors modulates the control of left/right sidedness in rat embryos.     

A comparison of the properties of the cardiac beta-adrenergic receptor adenylyl cyclase system in the rat and the marmoset monkey

1. A comparison was made between adrenergic receptor binding properties and catecholamine-stimulated adenylyl cyclase activity in cardiac membrane fractions from the rat and the marmoset monkey. 2. [125I]HEAT and [125I]ICYP were used to determine respectively, the alpha- and beta-adrenergic receptor binding in cardiac membrane fractions. 3. Greatest adrenergic receptor density and degree of specific binding was evident using membranes sedimenting between 6000 and 46,000 g. 4. In rat heart, the ratio of beta- to alpha-adrenergic receptors was 57:43, while for the marmoset this ratio was 92:8. 5. Basal, isoproterenol, sodium fluoride and forskolin-stimulated adenylyl cyclase activities in the rat and marmoset monkey were investigated in several different cardiac membrane fractions. 6. The highest-fold stimulation of adenylyl cyclase activity was present in membranes sedimenting between 0 and 500 g. 7. Adenylyl cyclase activities were higher in the marmoset heart membrane preparations, however the rat heart adenylyl cyclase exhibited greater sensitivity to isoproterenol; ED50 3.8 X 10(-7) M compared with 7.5 X 10(-7) M for the marmoset. 8. Differences between rat and marmoset catecholamine-sensitive adenylyl cyclase activity were apparent when a variety of adrenergic agonists and antagonists were tested. 9. In the marmoset but not the rat, adrenergic antagonists alone stimulated basal adenylyl cyclase activity. 10. Differences in the activation of cardiac adenylyl cyclase by GTP and GMP-PNP were also evident between the rat and the marmoset monkey, particularly with regard to basal and isoproterenol-stimulated activity.     

Alpah-adrenergic receptor modulation of beta-adrenergic, adenosine and prostaglandin E1 increased adenosine 3':5'-cyclic monophosphate levels in primary cultures of glia.

Beta-adrenergic agonists, adenosine and prostaglandin E1 increased the level of adenosine 3':5'-monophosphate (cAMP) in glial cultures prepared from rat cerebral cortical tissue. In addition to these physiological effectors, cholera toxin also increased cAMP levels in these cultures. The accumulation of cAMP in response to each of these agen-s, including cholera toxin, was partially blocked (50--80%) by simultaneous alpha-adrenergic receptor stimulation. Basal levels of cAMP were not affected by alpha-adrenergic agonists. These results indicate that in glia, alpha-adrenergic receptors may serve to modulate the level of cAMP which normally accumulates in response to a number of neurohumoral substances. The modulatory effect of alpha-adrenergic agents does not appear to reduce cAMP accumulation by activating phosphodiesterase since the effect was not blocked by a potent inhibitor of this enzymemthe results suggest that the modulatory effect of alpha-adrenergic receptor activation results from an interaction which takes place at some point in between adenylate cyclase-associated-membrane receptors and the enzymatic degradation of cAMP.     

Interaction of alpha and beta adrenergic stimulation on aortic ornithine decarboxylase activity

The interaction between beta and alpha adrenergic agonists on regulation of cockerel aortic ornithine decarboxylase (ODC) activity was examined. The beta adrenergic agonist isoproterenol both reduced basal aortic ODC activity and prevented induction of the decarboxylase by the alpha adrenergic agonist methoxamine. 3-Isobutyl-1- methylxanthine (IBMX) similarly reduced basal ODC activity and blocked induction of the enzyme by methoxamine. When given ten minutes before or after methoxamine, isoproterenol prevented aortic ODC induction, but not large sustained increases in blood pressure evoked by the alpha adrenergic agonist. In contrast, when injected three hours after methoxamine, isoproterenol had no effect on already elevated levels of enzyme activity. Addition of isoproterenol (10(-7)M), IBMX (1 mM) or dibutyryl cAMP (2.5 mM) to isolated aortic segments cultured in minimal salts-glucose media evoked decreases in basal levels of ODC activity resembling those observed in the intact animal. These results suggest that the balance between alpha and beta adrenergic stimulation may be an important feature of the regulation of polyamine biosynthesis in artery wall cells.     

Effect of adrenergic agents on alpha-amylase release and adenosine 3',5'-monophosphate accumulation in rat parotid tissue slices.

The role of cyclic AMP in stimulus-secretion coupling with investigated in rat parotid tissue slices in vitro. Isoproterenol and norepinephrine stimulated a rapid intracellular accumulation of cyclic AMP, which reached a maximum level of 20-30 times the control value by 5 to 10 min after addition of the drug. Isoproterenol was approximately ten times more potent in stimulating both alpha-amylase release and cyclic AMP accumulation than were norepinephrine and epinephrine, which had nearly equal effects on these two parameters. Salbutamol and phenylephrine were less effectivema parallel order of potency and sensitivity was observed for the stimulation of adenylate cyclase activity in a washed particulate fractionmthe results suggest that these drugs are acting on a parotid acinar cell through a beta1-adrenergic mechanismmat the lowest concentrations tested, each of the adrenergic agonists stimulated significant alpha-anylase release with no detectable stimulation of cyclic AMP accumulationmeven in the presence of theophylline, phenylephrine at several concentrations increased alpha-amylase release without a detectable increase in cyclic AMP levels. However, phenylephrine did stimulate adenylate cyclase. These data suggest that, under certain conditions, large increases in the intra-cellular concentration of cyclic AMP may not be necessary for stimulation of alpha-amylase release by adrenergic agonists. Also consistent with this idea was the observation that stimulation of cyclic AMP accumulation by isoproterenol was much more sensitive to inhibition by propranolol than was the stimulation of alpha-amylase release by isoproterenol. Stimulation of alpha-amylase release by phenylephrine was only partially blocked by either alpha- or beta-adrenergic blocking agents, whereas stimulation of adenylate cyclase by phenylephrine was blocked by propranolol and not by phentolaminemphenoxybenzamine and phentolamine potentiated the effects of norepinephrine and isoproterenol on both cyclic AMP accumulation and alpha-amylase release by N-6,O-2'-dibutyryl adenosine 3',5'-monophosphate; These observations may indicate a non-specific action of phenoxybenzamine, and demonstrate the need for caution in interpreting evidence obtained using alpha-adrenergic blocking agents as tools for investigation of alpha- and beta-adrenergic antagonism.     

High affinity binding of reovirus type 3 to cells that lack beta adrenergic receptor activity

A previous report demonstrated both immunological crossreactivity and structural similarity between the mammalian beta adrenergic receptor and the cell surface receptor for the reovirus type 3 (14). We now demonstrate that reovirus type 3 can bind selectively and with high affinity to cells that lack beta adrenergic receptor activity (L-cells). The present study was also designed to determine what effect reovirus binding has on beta adrenergic receptor function in cells (DDT1) that possess an intact ligand binding site. Based on computer analysis of reovirus competitive inhibition curves, the apparent dissociation binding constants (Kd) for reovirus binding to DDT1 and L-cells are 0.1 nM and 0.25 nM, respectively. High affinity [125I]-iodocyanopindolol (CYP) binding to beta adrenergic receptors can also be demonstrated in DDT1 cells but not in L-cells. In agreement with these ligand binding studies, adenylate cyclase activity is stimulated by the beta receptor agonist isoproterenol in DDT1 cell membranes but not in L-cell membranes. In addition, isoproterenol increases cAMP levels in DDT1 cells but not in L-cells. Neither reovirus serotype stimulates cAMP levels in either cell line, nor do they influence beta-adrenergic agonist stimulation of cAMP in DDT1 cells. These results argue against identity of the receptors for reovirus type 3 and beta adrenergic ligands.     

Alteration of sensitivity of adrenergic vascular responses after prolonged exposure to agonists via osmotic minipump

A study was performed to determine whether a constant 1-week exposure to either alpha or beta agonists in vivo would allow alteration or manipulation of the responses of rat aortic alpha- and beta-adrenergic receptors. Osmotic minipumps delivering either phenylephrine, isoproterenol, or propranolol for 7 days at a dose of 3.2, 4.2, or 5.2 mg/kg/day, respectively, were implanted in male Holtzman rats under halothane anesthesia. Seven days later, rats were killed and aortic ring preparations were used to measure alpha- and beta-adrenergic responses. In phenylephrine-pretreated rats, alpha-adrenergic responses, as measured by contractions induced by phenylephrine, were markedly reduced (P less than 0.05) across a dose range of 10(-9) to 10(-6) M. In contrast, in these same phenylephrine-pretreated preparations, the beta-adrenergic responses involving isoproterenol-induced relaxation were significantly increased (P less than 0.05) across a dose range of 10(-7) to 10(-5) M. Isoproterenol pretreatment for 7 days resulted in a statistically significant reduction of beta-adrenergic aortic relaxation, whereas the alpha-adrenergic responses to phenylephrine remained unchanged compared with controls. Propranolol pretreatment had no effect on either alpha- or beta-adrenergic responses. These findings indicate that the alpha agonist-induced response after in vivo pretreatment induces reciprocal changes in the functionally related beta-adrenergic apparatus, and also suggest linkage between these two receptors. In contrast, the beta response appears to desensitize or downregulate in response to beta agonist exposure in a manner that seems to be independent of or to operate in the absence of an alteration of the alpha response.     

Beta-adrenergic potentiation of endoplasmic reticulum Ca(2+) release in brown fat cells

We find that the adrenergic agonist isoproterenol increases intracellular Ca(2+) concentration ([Ca(2+)](i)) in cultured rat brown adipocytes. At the concentration used (10 microM), isoproterenol-induced Ca(2+) responses were sensitive to block by either alpha(1)- or beta-adrenergic antagonists, suggesting an interaction between these receptor subtypes. Despite reliance on beta-adrenoceptor activation, the Ca(2+) response was not due solely to increases in cAMP because, administered alone, the selective beta(3)-adrenergic agonist BRL-37344 or forskolin did not increase [Ca(2+)](i). However, increased cAMP elicited vigorous [Ca(2+)](i) increases in the presence of barely active concentrations of the alpha-adrenergic agonist phenylephrine or the P2Y receptor agonist UTP. Consistent with isoproterenol recruiting only inositol 1,4,5-trisphosphate (IP(3))-sensitive Ca(2+) stores, endoplasmic reticulum store depletion by thapsigargin blocked isoproterenol-induced Ca(2+) increases, but removal of external Ca(2+) did not. These results argue that increases in cAMP sensitize the IP(3)-mediated Ca(2+) release system in brown adipocytes.