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.     

Inhibition of lipolysis by adenosine is potentiated with age.

The ability of a variety of hormones to activate cells declines with age. We have investigated the mechanism for the reduced ability of beta adrenergic stimulation to activate lipolysis in fat cells from older rats. Previously, we have found that these cells have an intact lipolytic response to a cAMP analogue but diminished cAMP accumulation after isoproterenol stimulation, suggesting that the blunted cAMP response is rate limiting. In the present study we have tested the hypothesis that enhanced inhibition of lipolysis by endogenously released adenosine accounts for the diminished lipolysis. Adenosine deaminase was added to media containing the adipocytes from older rats to remove endogenous adenosine. Under these conditions beta adrenergic stimulation of lipolysis is intact in fat cells from older rats. The adenosine analogue N6-phenylisopropyladenosine more effectively inhibited lipolysis in the older group (77 +/- 6%) than in the younger group (46 +/- 5%), suggesting that enhanced efficacy of endogenous adenosine may account for the reduced lipolytic response to catecholamines. When pertussis vaccine was used to functionally inactivate adenosine receptors in adipocytes from the younger and older rats, the ability of isoproterenol to activate lipolysis was restored in the older group. All the data are consistent with the hypothesis that enhanced inhibitory effects of adenosine explain the diminished ability of beta adrenergic agonists to activate lipolysis. It is possible that enhanced inhibitory pathways may be involved in blunting responses to stimulatory hormones in other tissues from older animals.     

Activation of heat shock protein (hsp)70 and proto-oncogene expression by alpha1 adrenergic agonist in rat aorta with age.

Induction of heat shock proteins (hsp) most likely is a homeostatic mechanism in response to metabolic and environmental insults. We have investigated signal transduction mechanisms involved in alpha1, adrenergic receptor stimulation of hsp7O gene expression in isolated aortas with age. We found that alpha1 adrenergic agonists directly induced hsp70 mRNA in rat aorta in vitro; the alpha1, selective antagonist prazosin blocked this effect whereas chloroethylclonidine, an antagonist which has some selectivity for alpha1B receptors, was ineffective. This response was insensitive to pertussis toxin and was partially blocked by the protein kinase C inhibitor H7. Removal of extracellular calcium attenuated induction of hsp70 mRNA but not the induction of c-fos or c-myc. The induction of hsp70 mRNA by either norepinephrine or by phorbol dibutyrate was blunted in aortas from old (24-27 mo) rats whereas c-fos responses were not diminished in the older vessels. The hsp70 response to elevated temperature (42 degrees C) was not changed with age. Activation of hsp70 expression most likely involves a pertussis toxin insensitive G protein which activates protein kinase C, and requires extracellular calcium. With age, hsp70 gene expression induced by stimulation of alpha1 adrenergic receptors is markedly attenuated, which could modify responses to stress or vascular injury with aging.     

Albuterol-induced downregulation of Gsalpha accounts for pulmonary beta(2)-adrenoceptor desensitization in vivo

The aim of the present study was to develop a chronic in vivo model of pulmonary beta(2)-adrenoceptor desensitization and to elucidate the nature and molecular basis of this state. Subcutaneous infusion of rats with albuterol for 7 days compromised the ability of albuterol, given acutely, to protect against acetylcholine-induced bronchoconstriction. The bronchoprotective effect of prostaglandin E(2), but not forskolin, was also impaired, indicating that the desensitization was heterologous and that the primary defect in signaling was upstream of adenylyl cyclase. beta(2)-Adrenoceptor density was reduced in lung membranes harvested from albuterol-treated animals, and this was associated with impaired albuterol-induced cyclic adenosine monophosphate (cAMP) accumulation and activation of cAMP-dependent protein kinase ex vivo. Gsalpha expression was reduced in the lung and tracheae of albuterol-treated rats, and cholera toxin-induced cAMP accumulation was blunted. Chronic treatment of rats with albuterol also increased cAMP phosphodiesterase activity and G protein-coupled receptor kinase-2, but the extent to which these events contributed to beta(2)-adrenoceptor desensitization was unclear given that forskolin was active in both groups of animals and that desensitization was heterologous. Collectively, these results indicate that albuterol effects heterologous desensitization of pulmonary Gs-coupled receptors in this model, with downregulation of Gsalpha representing a primary molecular etiology.     

Subtype-selective regulation of beta adrenergic receptor-adenylyl cyclase coupling by phorbol esters in 3T3-L1 fibroblasts

To study the epigenetic regulation of beta adrenergic receptor subtypes, we examined the effects of phorbol esters on beta adrenergic receptor coupling to adenylyl cyclase in 3T3-L1 fibroblasts, which express both beta-1 and beta-2 adrenergic receptor subtypes. Pretreatment of intact 3T3-L1 cells with the protein kinase C activator phorbol dibutyrate caused a dose- and time-dependent decrease in subsequent cyclic AMP (cAMP) accumulation mediated by the beta adrenergic agonist isoproterenol. This effect was selective for beta-adrenergic receptor-mediated responses because there was a potentiation of cAMP accumulation caused by other activators such as prostaglandin E1, forskolin or cholera toxin. The inactive phorbol, alpha-phorbol dibutyrate was ineffective at 1 microM in attenuating isoproterenol stimulation, and 25 nM of the protein kinase C inhibitor staurosporine blocked the effects of phorbol ester on beta adrenergic agonist responses. Stimulation of cAMP accumulation by isoproterenol occurred through a greater proportion of beta-2 adrenergic receptors in phorbol dibutyrate-treated cells than in control cells. This was demonstrated using the beta-1 adrenergic selective antagonist ICI 89.406 and the beta-2 adrenergic selective antagonist ICI 118.551 to inhibit competitively isoproterenol-stimulated cAMP accumulation. Beta-2 adrenergic receptor number and subtype in these cells are regulated by glucocorticoids and butyrate. Decreasing the proportion of beta-1 adrenergic receptors and concomitantly increasing beta-2 adrenergic receptors with either glucocorticoids or butyrate decreased the ability of phorbol ester pretreatment to attenuate cAMP accumulation by isoproterenol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presynaptic modulation of beta adrenergic receptors in rat cerebral cortex after treatment with antidepressants.

Treatment with desmethylimipramine (DMI), a tricyclic antidepressant, for 7 to 21 days resulted in a 35 to 45% decrease in the accumulation of adenosine cyclic 3':5'-monophosphate (cAMP) in response to a maximally effective concentration of (-)-isoproterenol (ISO) in rat cerebral cortical slices. The EC50 for ISO-stimulated cAMP accumulation was not affected by DMI administration. The diminution in responsiveness to catecholamines was accompanied by a 35 to 40% decrease in the density of beta adrenergic receptors as measured by the binding of [125I]iodohydroxybenzylpindolol. Decreases in ISO-sensitive cAMP accumulation and in beta adrenergic receptor density were temporally correlated, maximal decreases being observed within 5 to 7 days. Within 7 days after cessation of chronic DMI treatment ISO-stimulated cAMP accumulation and beta adrenergic receptor density returned to normal. The role of presynaptic nerve terminals in mediating these phenomena was also investigated. Treatment of newborn rats with 6--hydroxydopamine inhibited the development of noradrenergic nerve terminals in the cerebral cortex and blocked the effects of DMI on cortical cAMP accumulation and on beta adrenergic receptor density. The administration of the beta adrenergic receptor antagonist propranolol led to increases in maximal ISO-stimulated cAMP accumulations and beta adrenergic receptor density in the rat cerebral cortex. This increase was not affected by the simultaneous administration of propranolol and DMI. Thus, the effect of DMI appears to be mediated through an action of norepinephrine at beta adrenergic receptors. Chronic treatment with two other clinically effective antidepressants, pargyline and iprindole, led to effects similar to those observed with DMI administration. Pretreatment of neonates with 6-hydroxydopamine blocked the effect of iprindole on beta adrenergic receptors. Preincubation of cortical membranes with guanosinetriphosphate before determination of the density of beta adrenergic receptors had no effect on the decreased number of receptors had no effect on the decreased number of receptors seen in DMI-treated animals. These experiments suggest that antidepressants, acting presynaptically, increase the concentration of transmitter at noradrenergic synapses and induce a compensatory decrease in the density of beta adrenergic receptors.     

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.     

Effects of protein kinase inhibitor, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, on beta-2 adrenergic receptor activation and desensitization in intact human lymphocytes

To investigate the role of protein kinases in agonist-mediated beta-2 adrenergic receptor regulation, the effects of the protein kinase A and C inhibitor, H-7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine], on isoproterenol-induced beta adrenoceptor activation and desensitization have been studied in intact human lymphocytes. In the presence of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, H-7 potentiated 10(-8) to 10(-4) M isoproterenol or prostaglandin E1-induced cyclic AMP (cAMP) accumulation in a dose-dependent manner. We failed to observe any effect of H-7 on forskolin-induced cAMP accumulation. These effects of H-7 are probably not due to its inhibition of phosphodiesterase. In addition, whereas up to 10(-3) M H-7 had no beta adrenergic receptor blocking effect, preincubation of intact cells with 10(-3.5) M H-7 partially prevented 50 nM isoproterenol-induced beta-2 adrenergic receptor desensitization in terms of decreases in beta adrenoceptor density (maximum binding), isoproterenol-mediated cAMP responsiveness and high affinity receptor binding for agonist. Interestingly, 10(-3.5) M H-7 alone treated cells also showed an up-regulation of cell surface beta receptor density (maximum binding) and increased cAMP responsiveness to isoproterenol stimulation. The mechanisms are unclear. If these effects occur as a result of inhibition by H-7 of protein kinase A and/or C, it may suggest an important role of protein kinase A and/or C in agonist-induced beta-2 adrenergic receptor regulation.     

Alpha 1 adrenergic receptor-induced c-fos gene expression in rat aorta and cultured vascular smooth muscle cells.

While growth of blood vessels is important in hypertension, relatively little is known about the contribution of catecholamines. Using isolated rat aorta and cultured smooth muscle cells, we examined adrenergic stimulation of gene expression. Phenylephrine, a selective alpha 1 adrenergic receptor agonist, caused a rapid and transient increase in c-fos mRNA accumulation which was inhibited by prazosin, an alpha 1 receptor antagonist. Similarly, phenylephrine stimulated c-jun and c-myc mRNA accumulation. Chloroethyl-clonidine, a compound which irreversibly blocks alpha 1B receptors, completely blocked the phenylephrine-induced increase in c-fos mRNA. RNase protection experiments demonstrated that rat aorta prominently expressed mRNA for alpha 1B and alpha 1A/D receptors. Phenylephrine-induced c-fos mRNA was partially inhibited by H-7, a protein kinase C inhibitor, and by nifedipine, a Ca2+ channel blocker; these two compounds together had additive effects. In situ hybridization showed that expression of c-fos mRNA induced by phenylephrine was localized to aorta's medial layer. These results suggest that alpha 1 receptor-induced increase in c-fos mRNA in aorta is mediated by a chloroethyl-clonidine-sensitive receptor subtype signaling via increasing intracellular Ca2+ concentrations and activating protein kinase C.     

Agonist-related differences in the relationship between cAMP content and protein kinase activity in canine trachealis.

We investigated the relationships between relaxation, cyclic AMP (cAMP) accumulation and cAMP-dependent protein kinase (cAMP-PK) activity in canine tracheal smooth muscle. In time course and concentration-response studies, forskolin and isoproterenol elicited relaxation of isolated trachealis strips that was accompanied by an increase in cAMP content and an activation of cAMP-PK. Although these results were consistent with the proposal that cAMP is a second messenger mediating relaxation of airway smooth muscle, close inspection of the data revealed a discrepancy in the relationship between cAMP accumulation and relaxation. To induce equivalent degrees of tracheal relaxation, forskolin generated greater increments in cAMP accumulation than did isoproterenol. On the other hand, the activation state of cAMP-PK correlated reasonably well with relaxation regardless of which agonist was used. Further analysis of the data revealed that the apparent disparity between cAMP accumulation and relaxation could largely be explained at the level of the relationship between cAMP content and cAMP-PK activity: compared to isoproterenol, forskolin induced greater increases in cAMP accumulation to achieve the same activation state of cAMP-PK. These observations lend support to the proposal that in canine trachealis, various components of the cAMP/cAMP-PK cascade exist in distinct subcellular compartments such that not all of the cAMP generated in response to forskolin has access to its target enzyme, cAMP-PK.     

Beta-1 and beta-2 adrenoceptor-mediated responses in preparations of pulmonary artery and aorta from young and aged rats

Rat pulmonary artery contains both alpha adrenoceptors mediating contraction and beta-1 and beta-2 adrenoceptors mediating relaxation. Neither alpha nor beta adrenoceptor-mediated responses of this vessel to norepinephrine or epinephrine were potentiated by cocaine, despite evidence for the presence of some adrenergic nerves. Beta adrenoceptor-mediated relaxation of pulmonary artery, but not aorta, modulated alpha adrenoceptor-mediated contractions to epinephrine but not norepinephrine. Rat aorta also contains both beta-1 and beta-2 adrenoceptors mediating relaxation, in that Schild plots for atenolol (beta-1 selective antagonist) using a beta-1 selective agonist (norepinephrine) and a beta-2 selective agonist (fenoterol), respectively, were not superimposed. The Schild plot data for lCl 118,551 (beta-2 selective) indicated that the minor population (beta-1) was less important in aorta than in pulmonary artery. On preparations from 18-month-old rats, the maximum relaxation to isoproterenol (20.4%, aorta and 67.9%, pulmonary artery) was less than in preparations from young rats (79.8%, aorta and 96.9%, pulmonary artery), i.e., aging had reduced the beta adrenoceptor-mediated relaxation in both vessels, but particularly in aorta. Also, the negative log EC50 of fenoterol, but not of isoproterenol or norepinephrine, was less than in preparations from young rats. This could indicate that aging had affected beta-2 more than beta-1 adrenoceptor-mediated responses and may explain why aging depressed the maximum relaxation of aorta more than that of pulmonary artery.     

Activation of alpha-2 adrenergic receptors augments neurotransmitter-stimulated cyclic AMP accumulation in rat brain cerebral cortical slices

Norepinephrine-stimulated cyclic AMP (cAMP) accumulation in brain is mediated by both alpha and beta adrenergic receptors. The functional activity of these receptors can be differentiated by examining the response to isoproterenol alone and isoproterenol + 6-fluoronorepinephrine, and alpha adrenergic agonist. The present study was undertaken to define the pharmacological characteristics of the alpha adrenergic component associated with cAMP accumulation in brain. Using a prelabeling technique it was found that norepinephrine- or isoproterenol plus 6-fluoronorepinephrine-stimulated cAMP accumulation was only inhibited partially by an alpha-1 adrenergic receptor antagonist. In contrast, alpha-2 adrenergic receptor antagonists inhibited completely that portion of the response exceeding that produced by isoproterenol alone (alpha adrenergic augmentation). Furthermore, selective alpha-1 adrenergic agonists were incapable of enhancing the cAMP response to isoproterenol, whereas alpha-2 adrenergic agonists were active in this regard. Partial agonists for the alpha-2 adrenergic receptor were ineffective as augmentors. The data suggest that the alpha adrenergic component of the norepinephrine response in rat brain slices has characteristics of both alpha-1 and alpha-2 receptors, with the alpha-2 adrenergic component being a major contributor in this regard.     

Norepinephrine- and isoproterenol- induced changes in cardiac contractility and cyclic adenosine 3':5' -monophosphate levels during early development of the embryonic chick.

Changes in contractility and cyclic adenosine 3':5'-monophosphate (cAMP) levels in response to norepinephrine and isoproterenol were monitored in isolated 4-day-old (noninnervated) and 7-day-old (innervated) embryonic hearts to determine whether a relationship between beta adrenergic receptor, adenylate cyclase and altered cardiac function is established at a very early stage in embryonic development before innervation takes place. Norepinephrine and isoproterenol promoted rapid time- and dose-dependent rises in cAMP levels which were greater in the 4-day-old hearts. These increases paralleled observed functional alterations within a specific range of drug concentrations and time. The elevation of cAMP levels and effect on cardiac function produced by isoproterenol (10(-7)M) were blocked by propranolol (10(-6)M). Dissociations between changes in tissue cAMP levels and cardiac function were also uncovered. Maximal increases in contractility were achieved with lower drug concentrations than were required to promote maximal accumulation of cAMP. Relatively high concentrations of norepinephrine or isoproterenol were less effective than lower concentrations in stimulating contractility but were more effective in promoting cAMP accumulation, The results indicate that both cardiac beta receptors adenylate cyclase are present and functionally related early in embryogenesis before sympathetic innervation occurs and that cAMP accumulation is associated with modulation of contractile activity whithin a certain concentration range and time course.     

Expression of the third intracellular loop of the delta-opioid receptor inhibits signaling by opioid receptors and other G protein-coupled receptors

To explore the feasibility of developing inhibitors of signaling by opioid receptors and other G protein-coupled receptors (GPCRs) that use the same G protein pool, we investigated the capacity of a minigene encoding the third intracellular loop of the delta-opioid receptor (delta-i3L) to act as competitive antagonist of the receptor-G protein interface interaction. In delta-i3L-expressing cells, the peptide blocked high-affinity agonist binding to both the delta- and the mu-opioid (delta-OR and mu-OR) and attenuated opioid and alpha2-adrenergic receptor (alpha2AR)-dependent [35S]guanosine-5'-O-(3-thio)triphosphate binding. Furthermore, delta-i3L expression resulted in inhibition of delta-, mu-OR-, and alpha2AR-receptor-mediated cAMP accumulation, whereas the cAMP response produced by activation of the beta2-adrenergic receptor was unaffected, suggesting that the inhibitory effects of delta-i3L expression were selective for Gi/Go proteins. Moreover, although delta-i3L expression also attenuated drastically phospholipase C accumulation and Ca2+ release following mu- and delta-OR stimulation, it failed to inhibit carbachol-mediated stimulation of inositol phosphate accumulation in M1-muscarinic receptor-expressing human embryonic kidney 293 cells. Finally, we also examined the effects of delta-i3L expression on the regulation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase pathway. Our results demonstrate that, although ERK activation by mu- and delta-ORs is attenuated by the presence of delta-i3L, ERK activation mediated by alpha2AR remained unaffected. Collectively, our data demonstrate that the delta-i3L can be used as potent inhibitor of G protein signaling for various GPCRs that use a common pool of G proteins.     

Characterization of a bromoacetylated derivative of pindolol as a high affinity, irreversible beta adrenergic antagonist in cultured cells

We have utilized cultured cell lines to test the utility of N8-(bromoacetyl)-N1-[3-(4-indolyoxy)-2-hydroxypropyl]-(Z)-1,8-diam ino- p-menthane, BIM, a recently synthesized, irreversible beta adrenergic antagonist. Previously available irreversible antagonists of beta adrenergic receptors have generally exhibited low affinity (typical IC50 values greater than or equal to 1 microM). By contrast, S49 lymphoma cells incubated with 10 nM BIM for 120 min and then washed extensively showed a 70% loss in beta adrenergic receptors, as measured by [125I]iodocyanopindolol binding. This loss, which could be prevented by propranolol, represented a decrease in receptor number without a change in affinity of the remaining receptors for [125I]iodocyanopindolol. The BIM-induced decrease in binding sites was persistent in membranes incubated for several hours after BIM treatment. BIM did not inactivate alpha-1 adrenergic receptors on Madin Darby canine kidney cells, alpha-2 adrenergic receptors on human erythroleukemia cells, nor did BIM treatment alter guanyl-5'-yl-imidodiphosphate-mediated regulation of agonist binding to the beta adrenergic receptors in S49 cell membranes. BIM treatment decreased cyclic AMP (cAMP) accumulation in S49 cells in response to the beta adrenergic agonist isoproterenol, but increased prostaglandin E1-stimulated cAMP accumulation (P = .09) without altering cAMP production in response to forskolin. The inactivation of beta receptors in S49 cells by BIM (IC50 = 0.30 nM) correlated closely with the loss in beta adrenergic receptor-mediated cAMP accumulation in these cells (IC50 = 0.59 nM), implying the absence of substantial receptor reserve for this response. We conclude that BIM is a potent, irreversible, selective beta adrenergic antagonist for the study of beta adrenergic receptors in cultured cells.     

Prostanoid receptors regulate the volume-sensitive efflux of osmolytes from murine fibroblasts via a cyclic AMP-dependent mechanism

The ability of prostanoid receptors to regulate the volume-dependent efflux of the organic osmolyte taurine from murine fibroblasts (L cells) via a cAMP-dependent mechanism has been examined. Incubation of L cells under hypoosmotic conditions resulted in a time-dependent efflux of taurine, the threshold of release occurring at 250 mOsM. Addition of prostaglandin E(1) (PGE(1)) potently (EC(50) = 2.5 nM) enhanced the volume-dependent efflux of taurine at all time points examined and increased the threshold for osmolyte release to 290 mOsM. Maximal PGE(1) stimulation (250-300% of basal) of taurine release was observed at 250 mOsM. Of the PGE analogs tested, only the EP(2)-selective agonist butaprost (9-oxo-11alpha,16S-dihydroxy-17-cyclobutyl-prost-13E-en-1-oic acid) was able to enhance taurine efflux. Inclusion of 1,9-dideoxyfoskolin, 5-nitro-2-(3-phenylpropylamino) benzoic acid, or 4-[(2-butyl-6,7-dicloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]-butanoic acid blocked the ability of PGE(1) to enhance taurine release, indicating the mediation of a volume-sensitive organic osmolyte and anion channel. The ability of PGE(1) to increase osmolyte release from L cells was mimicked by the addition of agents that inhibit cAMP breakdown, directly activate adenylyl cyclase, or are cell-permeant analogs of cAMP. Taurine release elicited by either PGE(1) or 8-(4-chlorophenylthio)-cAMP was attenuated by >70% in L cells that had been stably transfected with a mutant regulatory subunit of cAMP-dependent protein kinase (PKA). PGE(1) stimulation of taurine efflux was not attenuated by either depletion of intracellular calcium or inhibition of protein kinase C. The results indicate that activation of prostanoid receptors on murine fibroblasts enhances osmolyte release via a cAMP and PKA-dependent mechanism.     

Effect of dietary lipids on inotropic responses of isolated rat left atrium: attenuation of maximal responses by an unsaturated fat diet

The effect of varying the dietary fatty acid composition on inotropic responses to various pharmacological agents was investigated in isolated rat left atrium. Pregnant rats were fed either semisynthetic diets supplemented with coconut oil (saturated fatty acids), sunflower oil (unsaturated) or Purina Rodent Chow. Newborns were exposed through the maternal milk and later fed the same diets throughout adulthood. Sunflower oil caused a significant decrease in the maximal response of adult atria to norepinephrine, epinephrine and isoproterenol compared with the other diets. However, no differences in contractile response to norepinephrine were detected at ages 11 and 30 days, indicating a delayed onset of the response changes. We had previously demonstrated defects in the beta adrenoceptor-adenylate cyclase system in homogenates of atria from adult rats fed sunflower oil that may partly explain the attenuated adrenergic response. Additional inotropic studies were performed to further examine the role of this system. There was no change in the maximal contractile response to agents acting through cyclic AMP [cAMP (adenosine 3',5'-cyclic monophosphate)]-independent mechanisms, calcium and phenylephrine. In contrast, maximal responses to forskolin, 3-isobutyl-1-methylxanthine, and N6,2'-O-dibutyryl cAMP, which act via cAMP-dependent mechanisms, were significantly depressed by dietary sunflower oil. No differences were detected in cAMP hydrolysis by phosphodiesterase. These data are consistent with the hypothesis that alterations in adrenergic responsiveness of rat atria after dietary lipid treatment involve functional changes in the adenylate cyclase pathway distal to the enzyme.     

Forskolin Stimulates Aggrecan Gene Expression in Cultured Bovine Chondrocytes

Prostaglandins are autacoids that elevate intracellular 3prime prime or minute:5prime prime or minute-cyclic adenosine monophosphate (cAMP) levels in chondrocytes and other cells in culture. To facilitate intracellular cAMP accumulation, bovine chondrocytes were incubated with forskolin alone or forskolin and isobutylmethylxanthine. Both significantly increased proteoglycan synthesis, which was inhibited by the cAMP-dependent protein kinase inhibitor H89. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) on 3--16% gels revealed the presence of two large proteoglycan core proteins which migrated more slowly than the 200-kDa marker protein and two small proteoglycan core proteins which migrated slightly slower than the 46-kDa marker. Northern blot hybridization, employing (32)P-labeled cDNA probes, showed that aggrecan steady-state mRNA levels were increased by forskolin and isobutylmethylxanthine after 1 h and 5 h incubation. Decorin and type II collagen mRNA levels were not altered under these conditions. Link protein mRNA levels were slightly elevated, but only at the 5-h time point. These results indicated that stimulation of intracellular cAMP accumulation by forskolin or forskolin and isobutylmethylxanthine resulted in augmented proteoglycan synthesis via increased steady-state aggrecan mRNA levels. Suppression of proteoglycan synthesis by the cAMP-dependent protein kinase inhibitor H89 suggested that cAMP-dependent protein kinase may also play a role in regulating the synthesis and completion of newly synthesized proteoglycans.     

Decreased vascular relaxation in hypertension.

Relaxation of spirally cut aortic strips was diminished in vessels from both spontaneously hypertensive rats and renal hypertensive rats. Aortic relaxation was decreased in response to the cyclic nucleotides and the beta adrenergic stimulant, isoproterenol, in both models, of hypertension. Defective aortic relaxation also occurred with two other vasodilators, nitroglycerin and adenosine. Further evidence for a reduced relaxant ability of blood vessels from hypertensive rats was obtained by measuring aortic relaxation after exposure and subsequent removal of vascular contractile agonists. The time for aortic preparations from spontaneously hypertensive rats to relax to base-line tension after maximum contraction with norepinephrine, serotonin and potassium chloride was significantly prolonged compared to recovery time for vessels from Kyoto Wistar normotensive rats. Treatment of the spontaneously hypertensive rat with reserpine, but not hydralazine, resulted in an improved ability of aortic preparations to relax. Based on these data, we propose that defects in vascular relaxation may contribute to hypertension and that some antihypertensive drugs may improve or facilitate vascular relaxation.     

Inhibition of the low km cyclic AMP phosphodiesterase in intact canine trachealis by SK&F 94836: mechanical and biochemical responses

The mechanical and biochemical responses of the canine trachealis to SK&F 94836 [2-cyano-1-methyl-3-[4-(4-methyl-6-oxo- 1,4,5,6-tetrahydropyridazine-3-yl)phenyl]guanidine], a selective inhibitor (ki = 1-3 microM) of the low km cyclic AMP (cAMP) phosphodiesterase, were assessed. Time course studies indicated that SK&F 94836-induced relaxation of trachealis strips contracted with 0.1 microM methacholine was accompanied by an activation of cAMP-dependent protein kinase (cAMP-PK). In subsequent experiments, trachealis strips were contracted with three concentrations of methacholine (0.1, 1.0 or 3.0 microM) or two concentrations of histamine (10 or 300 microM) before being relaxed by the cumulative addition of SK&F 94836. The relaxant response to SK&F 94836 (EC50 = 1-10 microM) decreased progressively as tissues were contracted with higher concentrations of methacholine. In parallel with its inhibitory effect on SK&F 94836-induced relaxation, methacholine suppressed the ability of SK&F 94836 to activate cAMP-PK. Interestingly, the inhibition of cAMP-PK activity was not accompanied by a significant inhibition of SK&F 94836-stimulated cAMP accumulation. Unlike the results with methacholine, the concentration of histamine used to contract tissues had no effect on SK&F 94836-induced relaxation or cAMP-PK activation. To determine the effect of SK&F 94836 on the mechanical and biochemical responses to the beta adrenoceptor agonist isoproterenol, tissues were first contracted with 3.0 microM methacholine and then incubated with 0, 0.3, 3.0 or 30 microM SK&F 94836 before being relaxed by the cumulative addition of isoproterenol. In these experiments, SK&F 94836 potentiated isoproterenol-induced relaxation, cAMP accumulation and cAMP-PK activation in a concentration-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)