Age-related changes in the control of hepatic cyclic AMP levels by alpha 1- and beta 2-adrenergic receptors in male rats

Hepatocytes from juvenile male rats (80-110 g) showed a 12-fold elevation of cAMP in response to epinephrine, which was mediated by beta 2-adrenergic receptors. In these cells, either alpha 1- or beta 2-adrenergic stimulation alone activated phosphorylase and glucose release although the alpha 1-phosphorylase response was 10-fold more sensitive to epinephrine and resulted in more rapid (by 10-20 s) activation of the enzyme. This suggests that the beta 2-adrenergic response is functionally unimportant for glycogenolysis, even in juvenile rats. beta 2-Adrenergic stimulation did, however, produce an increase in the rate of gluconeogenesis from [U-14C] lactate in these cells. Aging in the male rat was associated with attenuation of the beta 2-adrenergic cAMP response coupled with the emergence of an alpha 1-receptor-mediated accumulation of cAMP. The order of potency displayed by the alpha 1-adrenergic/cAMP system to adrenergic agonists and antagonists was identical with that of the alpha 1-adrenergic/Ca2+ system. These data suggest that, in maturity, hepatic alpha 1-receptors become linked to 2 separate transduction mechanisms, namely Ca2+ mobilization and cAMP generation. Calcium depletion of hepatocytes from adult, but not juvenile, male rats increased the alpha 1-component of the cAMP response to epinephrine, but under these conditions, alpha 1-activation of phosphorylase occurred more slowly than in calcium-replete cells. Blockade of alpha 2-adrenergic receptors did not significantly modify catecholamine effects on hepatocyte cAMP or phosphorylase a levels in male rats at any age studied, suggesting a lack of functional significance for these receptors in the regulation of glycogenolysis.     

Multiple transduction mechanisms are likely involved in calcium-mediated exocrine secretory events in rat parotid cells

The parotid gland of the aged rat provides an example of an altered alpha 1-adrenergic physiologic response (K+ efflux) resulting from a postreceptor perturbation in signal transduction mechanisms (Ito, H., Baum, B. J., Uchida, T., Hoopes, M. T., Bodner, L. & Roth, G. S. (1982) J. Biol. Chem. 257, 9532-9538). This alteration in gland function can be completely circumvented by eliciting K+ efflux via the Ca2+-ionophore, A23187, at several Ca2+ concentrations (ibid.). Since Ca2+ is purported to mediate other secretory events in the rat parotid, we have probed neurotransmitter regulated Ca2+ mobilization and secretory mechanisms in this tissue by employing an aging paradigm. The responses studied were alpha-adrenergic- and muscarinic-cholinergic-mediated K+ efflux, 45Ca2+ release, and amylase secretion. No differences were detected between young (3 months) and old (24 months) cell preparations for any muscarinic-cholinergic agonist-induced response studied. Following alpha-adrenergic stimulation, K+ efflux and 45Ca2+ release from old cell preparations were reduced markedly, while no changes were found for the amylase secretion response. These results suggest that 1) alpha-adrenergic and cholinergic signal transduction mechanisms for K+ efflux and 45Ca2+ release are dissociated in cells of the rat parotid gland, and 2) following alpha 1-adrenergic stimulation, signal transduction likely proceeds by at least two pathways, one which is apparently involved in protein excytosis (intact in cells from old rats) and the other which is apparently involved in K+ efflux and 45Ca2+ release (perturbed in old cells).     

Evidence for an alteration in the microsomal Ca2+ release mechanism in senescent rat parotid acinar cells

Previously, we have shown that Ca2+ mobilization following an alpha 1-adrenergic receptor stimulus is reduced in parotid acinar cells from senescent rats as a result of an altered ability of inositol 1,4,5-trisphosphate (IP3) to induce Ca2+ release from a non-mitochondrial, intracellular Ca2+ store (Ishikawa, Y., et al. Biochim. Biophys. Acta 968, 203-210). We have used this model to examine the IP3-induced Ca2+ release mechanism in these cells. 45Ca2+ efflux, after exposure to (-) epinephrine, from cells of young adult (3-6 months) rats was approx. 2-fold that observed from cells from older animals (approx. 24 months) either in the presence or absence of extracellular Ca2+. Similarly, cytosolic Ca2+ levels were greater in cells of young adult rats under these same incubation conditions. However, microsomal membrane preparations, from both age groups displayed similar IP3 binding sites (Kd approximately 90 nM, Bmax approximately 850 fmol/mg protein) and ATP-dependent Ca2+ transport ability (approx. 8 nmol/mg protein.min -1). These data suggest that there is an alteration in the IP3-induced Ca2+ release mechanism in microsomal membranes of parotid glands from senescent rats which may account for the decreased Ca2+ release seen after agonist stimulation of this tissue.     

Enhanced role for RhoA-associated kinase in adrenergic-mediated vasoconstriction in gracilis arteries from obese Zucker rats

Obesity, insulin resistance, dyslipidemia, and hypertension are components of the pathophysiological state known as metabolic syndrome. Adrenergic vasoconstriction is mediated through increases in cytosolic Ca2+ and the myofilaments' sensitivity to Ca2+. In many pathophysiological states, there is an enhanced role for Rho kinase (ROK)-mediated increases in Ca2+ sensitivity of the contractile apparatus. Thus we hypothesized that there is a greater role for ROK-mediated increases in Ca2+ sensitivity in alpha1-adrenergic vasoconstriction in arteries from obese Zucker (OZ) rats. Therefore, small gracilis muscle arteries from 11- to 12-wk-old and 16- to 18-wk-old lean and OZ rats were isolated, cannulated, and pressurized to 75 mmHg. For some experiments, vessels were loaded with fura 2-AM. Changes in luminal diameter and vessel wall Ca2+ concentration ([Ca2+]) were measured in response to phenylephrine (PE), the thromboxane mimetic U-46619, and KCl. alpha1-Adrenergic vasoconstriction was similar between 11- to 12-wk-old lean and obese animals and greater in older obese animals compared with controls. PE-induced increases in vascular smooth muscle cell [Ca2+] were blunted in OZ animals compared with lean controls in both age groups of animals. KCl and U-46619 elicited similar vasoconstriction and vascular smooth muscle cell [Ca2+] in both groups. ROK inhibition attenuated PE vasoconstriction to a greater degree in arteries from 11- to 12-wk-old OZ rats compared with lean animals; ROK inhibition in arteries from older rats right shifted both concentration-response curves to the same point. Total RhoA and ROKalpha protein expressions were similar between groups. These results suggest an enhanced role for the ROK pathway in alpha1-adrenergic vasoconstriction in metabolic syndrome.     

Alpha 1-adrenergic receptor activation mobilizes cellular Ca2+ in a muscle cell line

Regulation of cellular Ca2+ movements by alpha 1-adrenergic receptors has been studied using 45Ca2+ flux techniques in monolayer cultures of intact BC3H-1 cells. Unidirectional 45Ca2+ efflux from BC3H-1 cells reveals multiphasic kinetics, with a major fraction of cellular Ca2+ residing in a slowly exchanging intracellular compartment. Stimulation of alpha 1-adrenergic receptors by the agonist phenylephrine substantially increases 45Ca2+ unidirectional efflux, accompanied by a far smaller increase in 45Ca2+ influx. The selective enhancement of 45Ca2+ unidirectional efflux upon alpha 1-adrenergic receptor activation results in a net 30-40% decline in total cell Ca2+ content, measured either by radioisotopic equilibrium techniques or by atomic absorption spectroscopy. The relatively large pool of Ca2+ responsive to alpha-adrenergic stimulation is not displaced by La3+ but can be depleted with the Ca2+ ionophore A-23187. These results indicate that alpha 1-adrenergic receptor activation predominantly mobilizes Ca2+ from intracellular stores, together with a much smaller increase in transmembrane Ca2+ permeability. This interpretation is supported by comparative 45Ca2+ flux studies using a sister clone of BC3H-1 cells possessing surface nicotinic acetylcholine receptors but no alpha 1-adrenergic receptors. Agonist stimulation of the cholinergic receptor opens a well characterized transmembrane ion permeability gate. Cholinergic receptor activation greatly enhances the observed 45Ca2+ unidirectional influx relative to efflux, leading to net elevation of cellular Ca2+ content as Ca2+ moves down its inwardly directed concentration gradient.     

Presence of a Ca2+-dependent K+ channel in brown adipocytes. Possible role in maintenance of alpha 1-adrenergic stimulation

We have previously demonstrated mobilization of Ca2+ in and efflux of Rb+ (K+) from isolated hamster brown adipocytes as a consequence of norepinephrine stimulation. We have now investigated the adrenoceptor subtype specificity of these responses and found them both to be of the alpha 1-subtype. Further, we have found that the Rb+ (K+) efflux was dependent upon a primary Ca2+ mobilization event in response to the alpha 1-adrenergic stimulation, since the Rb+ efflux could also be demonstrated by the addition of the Ca2+ ionophore A23187 to the cells. The norepinephrine- and A23187-stimulated Rb+ effluxes were both inhibited by the Ca2+-dependent K+-channel blocker apamin. Apamin also significantly attenuated Ca2+ mobilization in cells in response to a submaximal concentration of norepinephrine. We conclude that alpha 1-adrenergic stimulation of brown fat cells leads to a mobilization of intracellular Ca2+ which, in itself or via other mechanisms, leads to an increase in cytosolic Ca2+ concentration which, in turn, activates a Ca2+-dependent K+ channel, leading to a K+ release from these cells. A possible role for this channel to sustain and augment the response to alpha 1-adrenergic stimulation is discussed.     

Role of a guanine nucleotide-binding protein in alpha 1-adrenergic receptor-mediated Ca2+ mobilization in DDT1 MF-2 cells

In this study the mechanisms involved in alpha 1-adrenergic receptor-mediated Ca2+ mobilization at the level of the plasma membrane were investigated. Stimulation of 45Ca2+ efflux from saponin-permeabilized DDT1 MF-2 cells was observed with the addition of either the alpha 1-adrenergic agonist phenylephrine and guanosine-5'-triphosphate or the nonhydrolyzable guanine nucleotide guanylyl-imidodiphosphate. In the presence of [32P]NAD, pertussis toxin was found to catalyze ADP-ribosylation of a Mr = 40,500 (n = 8) peptide in membranes prepared from DDT1 MF-2 cells, possibly the alpha-subunit of Ni. However, stimulation of unidirectional 45Ca2+ efflux by phenylephrine was not affected by previous treatment of cells with 100 ng/ml pertussis toxin. These data suggest that the putative guanine nucleotide-binding protein which couples the alpha 1-adrenergic receptor to Ca2+ mobilization in DDT1 MF-2 cells is not a pertussis toxin substrate and may possibly be an additional member of the guanine nucleotide binding protein family.     

Ca2+ influx mediates enhanced alpha2-adrenergic contraction in aortas from rats treated with NOS inhibitor

Previously, we reported that aortic segments from rats made hypertensive with the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (L-NNA) exhibit enhanced contractile sensitivity to both alpha2-adrenergic receptor (alpha2-AR) stimulation and to KCl-induced depolarization. We hypothesized that increased contractile responses to these agents was due to a change in the common effector L-type voltage-dependent calcium channel (VDCC). In aortic segments from control and L-NNA-treated rats, contraction to the alpha2-AR agonist UK-14304 stimulated Ca2+ influx but released intracellular Ca2+ only in control arteries. UK-14304-induced contraction was blocked by the VDCC antagonist nifedipine in both control and L-NNA aortas but contraction of aortas from L-NNA-treated rats was blocked by lower concentrations. Calcium imaging studies in fura 2-loaded freshly isolated aortic vascular smooth muscle cells also demonstrated UK-14304-stimulated Ca2+ influx sensitive to nifedipine only in cells from L-NNA-treated rats. We conclude that alpha2-AR contraction in the rat aorta is mediated primarily by Ca2+ influx and that L-NNA-induced hypertension increases the dependence of this contraction on VDCCs.     

Alpha 1-Adrenergic stimulation of Ca2+ mobilization without phosphorylase activation in hepatocytes from phosphorylase b kinase-deficient gsd/gsd rats.

Phenylephrine, vasopressin and the bivalent cation ionophore A23187 mobilized Ca2+ normally, but failed to activate phosphorylase, in hepatocytes from gsd/gsd rats with a deficiency of liver phosphorylase b kinase. These data provide strong evidence that phosphorylase b kinase is the site of action of the Ca2+ mobilized intracellularly during alpha 1-adrenergic activation of phosphorylase in liver cells.     

Alpha 1- and beta-adrenergic regulation of intracellular Ca2+ levels in brown adipocytes

In order to monitor changes in cytosolic Ca2+ levels, brown-fat cells were incubated with the fluorescent Ca2+-indicator fura-2 and the fluorescence intensity ratio followed. The addition of norepinephrine led to a rapid and persistent increase in the cytosolic Ca2+ level, which was dose-dependent with a maximal effect at about 1 microM. The response was diminished in the absence of extracellular Ca2+ and was inhibited more efficiently by phentolamine and prazosin than by propranolol or yohimbine, indicating alpha 1-adrenergic mediation. Accordingly, selective alpha 1-adrenergic stimulation also increased the cytosolic Ca2+ level. However, selective beta-adrenergic stimulation, as well as the adenylate cyclase activator forskolin, were also able to increase the cytosolic Ca2+ level in these cells to a certain extent. It was concluded that the major part of the increase in cytosolic Ca2+ was mediated, as in other cell types, via alpha 1-adrenergic receptors, but that Ca2+ levels were also positively modulated by a cAMP-mediated process. These observations are discussed in relation to known alpha 1/beta synergisms in brown adipose tissue.     

Effect of thyroid hormone on intracellular Ca2+ mobilization by noradrenaline and vasopressin in relation to glycogenolysis in rat liver

The relation between Ca2+ efflux, Ca2+ mobilization from mitochondria and glycogenolysis was studied in perfused euthyroid and hypothyroid rat livers stimulated by Ca2+-mobilizing hormones. Ca2+ efflux, induced by noradrenaline (1 microM) in the absence or presence of DL-propranolol (10 microM) from livers perfused with medium containing a low concentration of Ca2+ (approx. 24 microM), was decreased by more than 50% in hypothyroidism. This correlated with an equal decrease of the fractional mobilization of mitochondrial Ca2+, which could account for 65% of the difference between the net amounts of Ca2+ expelled from the euthyroid and hypothyroid livers. With vasopressin (10 nM) similar results were found, suggesting that hypothyroidism has a general effect on mobilization of internal Ca2+. In normal Ca2+ medium (1300 microM), however, the effect of vasopressin on net Ca2+ fluxes and phosphorylase activation was not impaired in hypothyroidism, indicating that Ca2+ mobilization from the mitochondria in this case plays a minor role in phosphorylase activation. The alpha 1-adrenergic responses of Ca2+ efflux, phosphorylase activation and glucose output, glucose-6-phosphatase activity and oxygen consumption in hypothyroid rat liver were completely restored by in vivo T3 injections (0.5 micrograms per 100 g body weight, daily during 3 days). Perfusion with T3 (100 pM) during 19 min did not influence hypothyroid rat liver oxygen consumption and alpha 1-receptor-mediated Ca2+ efflux. However, this in vitro T3 treatment showed a completely recovered alpha 1-adrenergic response of phosphorylase and a partly restored glucose-6-phosphatase activity and glucose output. The results indicate that thyroid hormones may control alpha 1-adrenergic stimulation of glycogenolysis by at least two mechanisms, i.e., a long-term action on Ca2+ mobilization, and a short-term action on separate stages of the glycogenolytic process.     

Changes in free cytosolic Ca2+ in hepatocytes following alpha 1-adrenergic stimulation. Studies on Quin-2-loaded hepatocytes

The Ca2+ selective fluorescent indicator, Quin-2, was employed to monitor continuously the concentration of free cytosolic Ca2+ [ Ca2+ ]i in isolated rat hepatocytes. Epinephrine (10(-6) M) and phenylephrine (10(-5) M), acting via alpha 1-adrenergic receptors, increases [ Ca2+ ]i from a basal concentration of approximately 0.2 microM to approximately 0.6 microM. This increase in [ Ca2+ ]i is evident as early as 1 to 1.5 s, the earliest time so far reported for any hepatic alpha 1-adrenergic event. Vasopressin (10(-8) M), after a lag which is 2 to 3 s longer, increases [ Ca2+ ]i to the same extent and at the same rate as the alpha 1-adrenergic agonists. Glucagon (10(-8) M) also increases [ Ca2+ ]i but at a significantly slower rate and only after a lag of about 10 s. All of these agents also induce an increase in the fluorescence of control cells. This Quin-2 independent fluorescence, which is due to an increased reduction of pyridine nucleotides, must be corrected for before the maximum change in [ Ca2+ ]i can be calculated but is sufficiently slow so as not to contribute to the initial rate of increase in the Quin-2-dependent fluorescence.     

Homologous and heterologous desensitization of one of the pathways of the alpha 1-adrenergic action. Effects of epinephrine, vasopressin, angiotensin II and phorbol 12-myristate 13-acetate

Activation of protein kinase C blocks the alpha 1-adrenergic action in hepatocytes. Preincubation of hepatocytes (in buffer with or without calcium) with vasopressin, angiotensin II, phorbol myristate acetate (PMA) or epinephrine + propranolol markedly diminished the alpha 1-adrenergic responsiveness of the cells (stimulation of ureagenesis) assayed in buffer without calcium. On the contrary, when the alpha 1-adrenergic responsiveness was assayed in buffer containing calcium no effect of the preincubation with vasopressin, angiotensin II or PMA was observed. Preincubation with epinephrine diminished the alpha 1-adrenergic responsiveness of the cells. In hepatocytes from hypothyroid rats the preincubation with the activators of protein kinase C (vasopressin, angiotensin II, phorbol 12-myristate 13-acetate and epinephrine) reduced markedly the alpha 1-adrenergic responsiveness of the cells, whereas in identical experiments using cells from adrenalectomized rats only the preincubation with epinephrine diminished the responsiveness. It is concluded that activation of protein kinase C induces desensitization of the alpha 1-adrenergic action in hepatocytes and that the calcium-independent pathway of the alpha 1-adrenergic action (predominant in cells from hypothyroid animals) resensitizes more slowly than the calcium-dependent pathway (predominant in cells from adrenalectomized rats). Epinephrine in addition to inducing this type of desensitization (through protein kinase C) leads to a further refractoriness of the cells towards alpha 1-adrenergic agonists.     

Alpha 2-adrenergic receptor stimulation mobilizes intracellular Ca2+ in human erythroleukemia cells

Human erythroleukemia cells are a model system for studies of alpha 2-adrenergic receptors and their coupling to inhibition of adenylate cyclase (McKernan, R. M., Howard, M. J., Motulsky, H. J., and Insel, P. A. (1987) Mol. Pharmacol. 32, 258-265). Using Fura-2, we show that alpha 2-adrenergic receptor stimulation also increases intracellular Ca2+ in these cells by 80-250 nM. Although epinephrine only inhibited forskolin-stimulated cAMP generation when beta-adrenergic receptors were blocked, the Ca2+ increase was not affected by beta-adrenergic receptor blockade. The Ca2+ increase was not affected by forskolin or 8-bromo-cAMP. Thus, alpha 2-adrenergic receptors independently couple to elevation of intracellular Ca2+ and adenylate cyclase inhibition. Chelating all extracellular Ca2+ did not reduce the response, demonstrating mobilization of intracellular, rather than influx of extracellular Ca2+. The epinephrine-stimulated Ca2+ mobilization occurred prior to any detectable increase in inositol-(1,4,5)-trisphosphate. It was abolished by pretreatment with pertussis toxin (which blocks some G protein-mediated processes), but not by aspirin and indomethacin (which inhibit cyclooxygenase), nordihydroguaiaretic acid (which inhibits lipoxygenase), or Na+-free buffer (to block any Na+H+ exchange). We conclude, therefore, that alpha 2-adrenergic receptors on human erythroleukemia cells couple to mobilization of intracellular Ca2+ via a (pertussis toxin-sensitive) G protein-mediated mechanism that is independent of inhibition of adenylate cyclase.     

Effect of adrenalectomy on Ca2+ signaling in rat hepatocytes

To pursue our studies of the effects of adrenalectomy on the adrenergic regulation of phosphorylase a, cAMP, cell calcium, and Ca2+ signaling in rat hepatocytes (Studer, R.K., and Borle, A.B. (1984) Biochim. Biophys. Acta 804, 377-385; Freudenrich, C.C., and Borle, A.B. (1988) J. Biol. Chem. 263, 8604-8610), we have further examined the alpha 1-adrenergic pathway in adrenalectomized and sham-operated male rats. We measured the number and affinity of alpha 1-adrenergic receptors, the cytosolic free Ca2+ concentration [(Ca2+]i) of hepatocytes with aequorin, inositol triphosphate (IP3) accumulation, and Ca2+ influx and efflux across the plasma membrane. We also compared the effects of vasopressin with those obtained with epinephrine. We found that the number of alpha 1-adrenergic receptors was slightly depressed (-23%), but that their affinity was unchanged. However, IP3 accumulation evoked by epinephrine was decreased 50%. This is probably the main cause for the depressed peak rise in [Ca2+]i we previously observed and reported. We also found that the basal resting Ca2+ influx was increased after adrenalectomy. Experiments with the beta-blocker propranolol, which abolished the epinephrine-evoked increase in Ca2+ influx, suggest that this effect may be mediated by cAMP, at least in adrenalectomized animals. The effects of vasopressin on IP3 [Ca2+]i and Ca2+ influx and efflux were also significantly decreased after adrenalectomy, indicating that alpha 1-adrenergic-mediated and other IP3-dependent Ca2+ signaling pathways are depressed after adrenalectomy.     

Effect of alpha-adrenergic blockers on calmodulin association with the nuclear matrix of rat liver cells during proliferative activation

A transient peak of cytosolic calmodulin (CaM) was produced during the prereplicative phase of rat liver cell proliferation following partial hepatectomy. After accumulating in the cytosol, CaM apparently translocated into the nuclei, associating with the nuclear matrix. The administration of alpha 1-adrenergic blockers to hepatectomized rats prevented the association of CaM with the nuclear matrix without affecting the increase in the total nuclear CaM. The inhibitory effect of the alpha 1-antagonists was reversed by the simultaneous injection of the alpha-agonist noradrenaline. Since the activation of alpha 1-adrenergic receptors results in the release of Ca2+ from endoplasmic reticulum stores, the results suggest that the association of CaM with the nuclear matrix during proliferative activation is mediated by Ca2+ released from endoplasmic reticulum and show that the association with the matrix is independent of its intranuclear accumulation.     

Activation of calcium mobilization and calcium influx by alpha 1-adrenergic receptors in a smooth muscle cell line

Alpha 1-adrenergic receptor (alpha 1R) mediated increases in the cytosolic levels of free Ca+2 and the inositol phosphates were measured in a smooth muscle cell line, DDT1. Norepinephrine (NE) stimulated a rapid increase in cytosolic Ca+2 by two distinct components: 1) release of Ca+2 from intracellular sites (mobilization), and 2) influx of extracellular Ca+2. The mobilization component was not affected by removal of extracellular Ca+2 or addition of La+3 or Co+2 to the buffer. The influx component was abolished by EGTA, La+3, or Co+2, but was not affected by the voltage-operated Ca+2 channel blockers diltiazem or nifedipine. Depolarization of DDT1 cells with 100 mM KCl or with gramicidin did not induce Ca+2 influx. NE also increased inositol trisphosphate to 78% over basal levels within 1 minute. These results suggest that alpha 1R on DDT1 cells are coupled to both the mobilization of intracellular Ca+2 and to receptor-operated Ca+2 channels in the plasma membrane, and that polyphosphoinositide hydrolysis may play a role in these phenomena.     

Phorbol Ester Pretreatment Desensitizes the Inhibition of Ca2+ Channels Induced by k-Opiate, α2-Adrenergic, and Muscarinic Receptor Agonists

Acute treatment of rat spinal cord-dorsal root ganglion cocultured neurons with 12-O-tetradecanoylphorbol 13-acetate (TPA), a known activator of protein kinase C, inhibited the dihydropyridine-sensitive voltage-dependent 45Ca2+ influx measured in these cells (IC50 of approximately 100 nM, 66% inhibition at 1 microM TPA). However, prolonged preincubation (24 h) of the cells with 100 nM TPA followed by extensive washing completely abolished, i.e., desensitized, the capacity of a second application of TPA to inhibit the activity of the voltage-dependent Ca2+ channels. Moreover, this treatment also abolished the inhibition of Ca2+ influx produced by kappa-opiate as well as by alpha 2-adrenergic and muscarinic receptor agonists. Substantial desensitization was already observed following a 1-h pretreatment with 100 nM TPA. In contrast to TPA, an inactive phorbol ester (4 beta-phorbol 13-acetate) did not affect the inhibition of the voltage-dependent Ca2+ influx by these receptor agonists. These results suggest that protein kinase C may have a role in the modulation of Ca2+ channels by kappa-opiate, alpha 2-adrenergic, and muscarinic receptor agonists.     

Aging-associated changes in whole cell K(+) and L-type Ca(2+) currents in rat ventricular myocytes

The effect of aging on cardiac membrane currents remains unclear. This study examined the inward rectifier K(+) current (I(K1)), the transient outward K(+) current (I(to)), and the L-type Ca(2+) channel current (I(Ca,L)) in ventricular myocytes isolated from young adult (6 mo) and aged (>27 mo) Fischer 344 rats using whole cell patch-clamp techniques. Along with an increase in the cell size and membrane capacitance, aged myocytes had the same magnitude of peak I(K1) with a greater slope conductance but displayed smaller steady-state I(K1). Aged myocytes also had a greater I(to) with an increased rate of activation, but the I(to) inactivation kinetics, steady-state inactivation, and responsiveness to L-phenylephrine, an alpha(1)-adrenergic agonist, were unaltered. The magnitude of peak I(Ca,L) in aged myocytes was decreased and accompanied by a slower inactivation, but the I(Ca,L) steady-state inactivation was unaltered. Action potential duration in aged myocytes was prolonged only at 90% of full repolarization (APD(90)) when compared with the action potential duration of young adult myocytes. Aged myocytes from Long-Evans rats showed similar changes in I(to) and I(Ca,L) but an increased I(K1). These results demonstrate aging-associated changes in action potential, in morphology, and in I(K1), I(to), and I(Ca,L) of rat ventricular myocytes that possibly contribute to the decreased cardiac function of aged hearts.     

alpha(1)-Adrenoceptor-induced Mg2+ extrusion from rat hepatocytes occurs via Na(+)-dependent transport mechanism

The stimulation of the alpha(1)-adrenergic receptor by phenylephrine results in a sizable extrusion of Mg2+ from liver cells. Phenylephrine-induced Mg2+ extrusion is almost completely abolished by the removal of extracellular Ca2+ or in the presence of SKF-96365, an inhibitor of capacitative Ca2+ entry. In contrast, Mg2+ extrusion is only partially inhibited by the Ca2+-channel blockers verapamil, nifedipine, or (+)BAY-K8644. Furthermore, Mg2+ extrusion is almost completely prevented by TMB-8 (a cell-permeant inhibitor of the inositol trisphosphate receptor), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (an intracellular Ca2+-chelating agent), or W-7 (a calmodulin inhibitor) Thapsigargin can mimic the effect of phenylephrine, and the coaddition of thapsigargin and phenylephrine does not result in an enlarged extrusion of Mg2+ from the hepatocytes. Regardless of the agonist used, Mg2+ extrusion is inhibited by >90% when hepatocytes are incubated in the presence of physiological Ca(2+) but in the absence of extracellular Na(+). Together, these data suggest that the stimulation of the hepatic alpha(1)-adrenergic receptor by phenylephrine results in an extrusion of Mg2+ through a Na(+)-dependent pathway and a Na(+)-independent pathway, both activated by changes in cellular Ca2+.