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.     

Simultaneous loss and reappearance of alpha 1-adrenergic responses and [3H]prazosin binding sites in rat liver after irreversible blockade by phenoxybenzamine

The relative influences of the in vivo administration of phenoxybenzamine on in vitro binding to alpha 1-adrenergic receptors and alpha 1-receptor-mediated responses were studied. Phenoxybenzamine treatment reduced maximal specific binding of the alpha 1-selective antagonist [3H]prazosin to liver cell membranes. This response was rapid (less than 90 min) and half-maximal following a phenoxybenzamine dose of approx. 10 mg/kg. A similar decrease in the ability of phenylephrine to stimulate glucose release and 45Ca2+ efflux from liver slices was also noted after phenoxybenzamine treatment. During the recovery period following administration of 30 mg/kg phenoxybenzamine, [3H]prazosin specific binding and phenylephrine-stimulated glucose release and 45Ca2+ efflux returned to their respective control levels with t 1/2 values of 42, 49 and 38 h, respectively. At all times studied during the recovery period, alpha 1-binding and both of the alpha 1-responses were similar fractions of their respective control values. These observations indicate that a close relationship exists between the density of [3H]prazosin binding sites and the ability of rat liver to respond to alpha 1-stimulation. We suggest that the binding sites identified in studies using the antagonist [3H]prazosin and those through which the agonist phenylephrine stimulates glucose release and 45Ca2+ efflux are either identical or in equilibrium with each other.     

Temporal integration of alpha 1-adrenergic responses in BC3H-1 muscle cells. Regulation of glycogen phosphorylase activity

Regulation of Ca2+-dependent glycogen phosphorylase activity by alpha 1-adrenergic and H1-histamine receptors has been examined in BC3H-1 muscle cells. Stimulation by either norepinephrine or histamine elevates the phosphorylase activity ratio within 5 s from a resting value of 0.37 +/- 0.03 to maximal values of 0.8-0.9. Phosphorylase activation by alpha-adrenergic agonists is sustained over 20-30 min of agonist exposure, whereas histamine exposure only transiently activates phosphorylase during the initial 5 min of stimulation. The initial activation of phosphorylase by either receptor is not attenuated by treated cells with Ca2+-deficient and [ethylenebis(oxyethylenenitrilo)]tetraacetic acid-supplemented buffer, whereas the response to sustained adrenergic stimulation depends largely, but not totally, upon extracellular Ca2+. The involvement of protein kinase C in agonist responses was tested by treating cells with phorbol 12-myristate 13-acetate. Phorbol 12-myristate 13-acetate inhibits receptor-mediated mobilization of intracellular Ca2+ (IC50 = 3.6 nM) yet activates phosphorylase independently of agonist. Phorbol 12-myristate 13-acetate has no effect on cellular 45Ca2+ fluxes in the absence of agonist. Thus, the two receptors coordinately regulate intracellular signaling through Ca2+- and protein kinase C-mediated pathways. alpha 1-Adrenergic receptors elicit sustained phosphorylase activation whereas H1-histaminergic receptors desensitize.     

Mobilization of intracellular calcium by alpha 1-adrenergic receptor activation in muscle cell monolayers

The fluorescent chelating agent quin 2 has been employed to monitor alterations of intracellular free Ca2+ concentrations ([Ca2+]i) in response to alpha 1-adrenergic receptor activation in adherent BC3H-1 cells. To correlate the kinetics of [Ca2+]i changes with transmembrane fluxes of this ion, continuous monitoring of [Ca2+]i has been undertaken on a monolayer of cells. Previous measurements of the transmembrane efflux of Ca2+ show a distinct lag in the response over a range of phenylephrine concentrations. By contrast, the elevation of [Ca2+]i is rapid (t1/2 approximately 2 s) and maintained for 30 s before it begins to decline to basal concentrations. The differences in kinetics indicate that the temporal delay in cellular Ca2+ efflux results from either activation of the transport system for Ca2+ extrusion or translocation of free Ca2+ to the transport site. The decline of [Ca2+]i with continued agonist exposure parallels both the efflux kinetics from the cell and the decline of total cellular Ca2+. At a time when free [Ca2+]i approaches the resting concentration, total cellular Ca2+ is reduced to a steady state value of 60% of that seen prior to stimulation. The Kact for phenylephrine-stimulated elevation in [Ca2+]i on the monolayer is 0.51 microM, which is similar to the Kact of 0.90 microM observed for phenylephrine-activated 45Ca2+ efflux. Addition of phentolamine subsequent to phenylephrine addition immediately reverses the agonist-stimulated Ca2+ mobilization, initiating a rapid return of [Ca2+]i to resting levels. A comparison of the kinetics of Ca2+ mobilization with its transmembrane flux suggests that the agonist augments the rate of recycling of intracellular Ca2+ between the free and bound states rather than causing release as a single bolus from the bound stores.     

Alpha 1-adrenergic responsiveness of young adult and aged rat submandibular cells in vitro

The present study has evaluated, in vitro, alpha 1-adrenergic receptor mediated responses in submandibular cells from young adult and aged rats. Submandibular glands from different aged rats possess a similar number of alpha 1-adrenergic receptors that display comparable binding characteristics. Following alpha 1-adrenergic stimulation, cells from both groups of rats show a similar ability to mobilize intracellular Ca2+ (45Ca2+ time course, agonist dose-response) and to elicit a functional response (inhibition of protein synthesis by epinephrine) which reflects Ca2+ mobilization.     

Cadmium evokes inositol polyphosphate formation and calcium mobilization. Evidence for a cell surface receptor that cadmium stimulates and zinc antagonizes

Cd2+ and other divalent metals mobilized cell Ca2+ in human skin fibroblasts. The divalent metals produced a large spike in cytosolic free Ca2+ and strikingly increased net Ca2+ efflux similarly to bradykinin. One-tenth microM Cd2+ half-maximally increased 45Ca2+ efflux. The potency order of the Ca2+ mobilizing metals was: Cd2+ greater than Co2+ greater than Ni2+ greater than Fe2+ greater than Mn2+. Cd2+ probably acts at an extracellular site because loading the cells with a heavy metal chelator only slightly inhibited Cd2+-evoked 45Ca2+ efflux. Cd2+ increased [3H]inositol polyphosphates; [3H]inositol trisphosphate increased 4-fold in 15 s. Zn2+ reversibly blocked 45Ca2+ efflux evoked by Cd2+ but not that produced by bradykinin. Zn2+ competitively (Ki = approximately 0.4 microM) inhibited net Ca2+ efflux produced by Cd2+. Cd2+ also evoked Ca2+ mobilization in umbilical artery muscle, endothelial, and neuroblastoma cells, and the divalent cation agonist and antagonist specificities were similar to those in the fibroblasts. The divalent metals appear to trigger Ca2+ mobilization via a reversible interaction with an external site on the cell surface, which may be considered a "Cd2+ receptor."     

Inhibition of alpha 1-adrenergic responsiveness in intact cells by a new, irreversible receptor antagonist

A novel alpha 1-adrenoreceptor antagonist, 1-(4-amino-6,7-dimethoxy-2-quinazolinyl)-4-(2-bicyclo [2.2.2] octa-2,5-dienylcarbonyl) piperazine, was synthesized and shown to potently block alpha 1-adrenoceptor-induced Ca2+ mobilization in intact rat parotid acinar cells. Irreversible inhibition was complete in less than 5 min. This alkylating prazosin derivative blocked Ca2+ release (IC50 approximately 5 X 10(-10)M) and [3H]-prazosin membrane binding (IC50 approximately 3 X 10(-10)M) in a concentration dependent fashion and increased the EC50 of epinephrine for Ca2+ efflux by approximately 35 fold. The agent however had no effect on muscarinic receptor-induced Ca2+ mobilization, or beta-adrenoreceptor-induced protein secretion, from cells. These findings suggest that this irreversible alpha 1-adrenoreceptor antagonist will be a valuable tool in probing alpha 1-adrenoreceptor function and metabolism in intact cells.     

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.     

Somatostatin: a metabolic regulator

Somatostatin, the hypothalamic release-inhibiting factor, has been found to stimulate gluconeogenesis in rat kidney cortical slices. Stimulation by somatostatin was linear and dose-dependent. Other bioactive peptides such as cholecystokinin, gastrointestinal peptide, secretin, neurotensin, vasoactive intestinal peptide, pancreatic polypeptide, beta endorphin and substance P did not affect the renal gluconeogenic activity. Somatostatin-induced gluconeogenesis was blocked by phentolamine (alpha adrenergic antagonist) and prazosin (alpha1 adrenergic antagonist) but not by propranolol (beta adrenergic antagonist) and yohimbine (alpha2 adrenergic antagonist) suggesting that the effect is via alpha1 adrenergic stimuli. Studies on the involvement of Ca2+ revealed that tissue depletion and omission of Ca2+ from the reaction mixture would abolish the stimulatory effect of somatostatin. Furthermore, somatostatin enhanced the uptake of 45calcium in renal cortical slices which could be blocked by lanthanum, an inhibitor of Ca2+ influx. It is proposed that the stimulatory effect of somatostatin on renal gluconeogenesis is mediated by alpha1 adrenergic receptors, or those which functionally resemble alpha1 receptors and that the increased influx of Ca2+ may be the causative factor for carrying out the stimulus.     

Rapid calcium mobilization by vasopressin and prostaglandin F2 alpha is independent of sodium influx in quiescent 3T3 cells

Vasopressin (VP) rapidly increased 45Ca2+ efflux. A VP antagonist prevented VP from mobilizing Ca2+ and stimulating DNA synthesis. Prostaglandin F2 alpha (PGF2 alpha) also stimulated rapid 45Ca2+ release. The effectiveness of different prostaglandins corresponded to their effectiveness as mitogens. The removal of external Na+ or Ca2+ had no effect on VP-or PGF2 alpha-induced 45Ca2+ release. The present results indicate that the mobilization of intracellular Ca2+ by these hormones is independent of Na+ or Ca2+ influx and that Ca2+ mobilization is important for growth stimulation.     

Vascular vasopressin receptors mediate phosphatidylinositol turnover and calcium efflux in an established smooth muscle cell line

Vasopressin-induced phosphatidylinositol turnover and mobilization of intracellular Ca2+ was studied using an established smooth muscle cell line (A-10). The cells were subcloned to ensure a monoclonal cell population. The accumulation of inositol mono-, di-, and tris-phosphates (IP1, IP2, and IP3, respectively), and the mobilization of intracellular Ca2+ were dependent on the time of incubation and the concentration of arginine vasopressin (AVP). IP1, IP2, and IP3 were significantly elevated after 15 sec and remained elevated for up to 2 hr. The concentrations of AVP required for half-maximal stimulation of IP1, IP2, and IP3 formation were 2, 12, and 4 nM, respectively. LiCl was required to observe the accumulation of inositol phosphates in response to AVP. Significant 45Ca2+ efflux was observed within 15 sec after exposure to AVP. By employing the vasopressin receptor subtype selective antagonists [d(CH2)5Tyr(Me)AVP, V1; d(CH2)5D-Tyr(Et)VAVP,V1/V2; d(CH2) 5D-IleVAVP,V2] and agonists [AVP, V1/V2; dDAVP, V2; dVDAVP, V2], we found that the vasopressin-induced stimulation of phosphatidylinositol turnover and 45Ca2+ efflux were mediated by receptors of the vascular V1 subtype. Pertussis toxin pretreatment partially inhibited vasopressin-induced phosphatidylinositol turnover. These data demonstrate that activation of V1 receptors of vascular smooth muscle cells resulted in enhanced phosphatidylinositol turnover and mobilization of intracellular Ca2+.     

Direct neurite-osteoblastic cell communication, as demonstrated by use of an in vitro co-culture system

Using an in vitro co-culture approach comprising cultured murine superior cervical ganglia and MC3T3-E1 osteoblast-like cells, we found that the addition of scorpion venom (SV) elicited neurite activation via intracellular Ca2+ mobilization and, after a lag period, osteoblastic Ca2+ mobilization. SV did not have any direct effect on the osteoblastic cells in the absence of neurites. The addition of an alpha1-adrenergic receptor (AR) antagonist, prazosin, dose-dependently prevented the osteoblastic activation that resulted as a consequence of neural activation by SV. These results demonstrate that osteoblastic activation occurred as a direct response to neuronal activation, which activation was mediated by alpha1-ARs in the osteoblastic cells.     

Calcium dependent aggregation of marine sponge cells is provoked by leukotriene B4 and inhibited by inhibitors of arachidonic acid oxidation

The mechanism of agonist-induced desensitization of the beta adrenergic receptor coupled adenylate cyclase has been studied in a smooth muscle cell line, BC3H-1, which expresses both alpha and beta adrenergic receptors and nicotinic receptors. beta receptors have been investigated in intact cells using as radioligand 3HCGP-12177, an hydrophilic compound which labels only surface receptors. The treatment of BC3H-1 cells with the agonist Isoproterenol, at 37 degrees but not at 4 degrees, induced a dose dependent internalization of the beta adrenergic receptor. Agonist-induced internalization was very rapid, in the order of few minutes. beta adrenergic receptor internalization was very specific: the alpha adrenergic agonist Phenylefrine had almost no effect on beta receptor levels, while Isoproterenol treatment had no effect on the number of alpha adrenergic or nicotinic receptors expressed at the cell surface of these cells. beta adrenergic receptor internalization is probably the major mechanism responsible for catecholamine desensitization in smooth muscle cells.     

Modulation of intracellular Ca2+ via L-type calcium channels in heart cells by the autoantibody directed against the second extracellular loop of the alpha1-adrenoceptors

The effects of methoxamine, a selective alpha1-adrenergic receptor agonist, and the autoantibody directed against the second extracellular loop of alpha1-adrenoceptors were studied on intracellular free Ca2+ levels using confocal microscopy and ionic currents using the whole-cell patch clamp technique in single cells of 10-day-old embryonic chick and 20-week-old fetal human hearts. We observed that like methoxamine, the autoantibody directed against the second extracellular loop of alpha1-adrenoreceptors significantly increased the L-type calcium current (I(Ca(L))) but had no effect on the T-type calcium current (I(Ca(T))), the delayed outward potassium current, or the fast sodium current. This effect of the autoantibody was prevented by a prestimulation of the receptors with methoxamine and vice versa. Moreover, treating the cells with prazosin, a selective alpha1-adrenergic receptor antagonist blocked the methoxamine and the autoantibody-induced increase in I(Ca(L)), respectively. In absence of prazosin, both methoxamine and the autoantibody showed a substantial enhancement in the frequency of cell contraction and that of the concomitant cytosolic and nuclear free Ca2+ variations. The subsequent addition of nifedipine, a specific L-type Ca2+ channel blocker, reversed not only the methoxamine or the autoantibody-induced effect but also completely abolished cell contraction. These results demonstrated that functional alpha1-adrenoceptors exist in both 10-day-old embryonic chick and 20-week-old human fetal hearts and that the autoantibody directed against the second extracellular loop of this type of receptors plays an important role in stimulating their activity via activation of L-type calcium channels. This loop seems to have a functional significance by being the target of alpha1-receptor agonists like methoxamine.     

Studies on the mechanism of inhibition of glucose-stimulated insulin secretion by noradrenaline in rat islets of Langerhans.

Noradrenaline (norepinephrine) was shown to be a potent inhibitor of glucose-induced insulin release from rat pancreatic islets, with half-maximal inhibition of the secretory response to 20 mM-glucose occurring at approx. 0.3 microM, and complete suppression of the response occurring at 4 microM-noradrenaline. Inhibition of insulin secretion by noradrenaline was antagonized by the alpha 2-adrenergic antagonist yohimbine (half maximally effective dose approximately 1 microM), but was largely unaffected by the alpha 1-adrenergic antagonist prazosin at concentrations up to 50 microM, suggesting that the response was mediated by alpha 2-adrenergic receptors. Noradrenaline significantly reduced the extent of 45Ca2+ accumulation in glucose-stimulated islets, although as much as 5 microM-noradrenaline was required for 50% inhibition of this response. The ability of noradrenaline to inhibit islet-cell 45Ca2+ uptake was totally abolished in media containing 1 mM-dibutyryl cyclic AMP, suggesting that the response may have been secondary to lowering of islet cyclic AMP. Under these conditions, however, noradrenaline was still able to inhibit insulin secretion maximally. The data suggest that the site(s) at which noradrenaline acts to mediate inhibition of insulin secretion in rat islets lies distal to both islet-cell cyclic AMP accumulation and Ca2+ uptake.     

Modulation of insulin release by adenosine A1 receptor agonists and antagonists in INS-1 cells: the possible contribution of 86Rb+ efflux and 45Ca2+ uptake

Due to the lack of specific agonists and antagonists the role of adenosine receptor subtypes with respect to their effect on the insulin secretory system is not well investigated. The A1 receptor may be linked to different 2nd messenger systems, i.e. cAMP, K+- and 45Ca2+ channel activity. Partial A1 receptor agonists are going to be developed in order to improve diabetes (increase in insulin sensitivity, lowering of FFA and triglycerides). In this study newly synthesized selective A1 receptor agonists and antagonists were investigated thereby integrating three parameters, insulin release (RIA), 45Ca2+ uptake and 86Rb+ efflux (surrogate for K+ efflux) of INS-1 cells, an insulin secretory cell line. The presence of A1-receptors was demonstrated by Western blotting. The receptor nonselective adenosine analogue NECA (5-N-ethylcarboxyamidoadenosine) at high concentration (10 microM) had no effect on insulin release and 45Ca2+ uptake which could be interpreted as the sum of effects mediated by mutual antagonistic adenosine receptor subtypes. However, an inhibitory effect mediated by A1 receptor agonism was detected at 10 nM NECA and could be confirmed by adding the A1 receptor antagonist PSB-36 (1-butyl-8-(3-noradamantyl)-3-(3-hydroxy-propyl)xanthine). NECA inhibited 86Rb+ efflux which, however, did not fit with the simultaneous inhibition of insulin secretion. The selective A1 receptor agonist CHA (N6-cyclohexyladenosine) inhibited insulin release; the simultaneously increased Ca2+ uptake (nifedipine dependent) and inhibition of 86Rb+ efflux did not fit the insulin release data. The CHA effect (even the maximum effect at 50 microM) can be increased by 10 microM NECA indicating that CHA and NECA have nonspecific and physiologically non-relevant effects on 86Rb+ efflux in addition to their A1-receptor interaction. Since PSB-36 did not influence the NECA-induced inhibition of 86Rb+ efflux, the NECA effect is not mediated by potassium channel-linked A1 receptors. The nonselective adenosine receptor antagonist caffeine increased insulin release which was reversed by CHA as expected when hypothesizing that both act via A1 receptors in this case. In conclusion, stimulation of A1 receptors by receptor selective and nonselective compounds reduced insulin release which is not coupled to opening of potassium channels (86Rb+ efflux experiments) or inhibition of calcium channels (45Ca2+ uptake experiments). It may be expected that of all pleiotropic 2nd messengers, the cAMP system (not tested here) is predominant for A1 receptor effects and the channel systems (K+ and Ca2+) are of minor importance and do not contribute to insulin release though being coupled to the receptor in other tissues.     

Mediation of Norepinephrine-Stimulated Cyclic AMP Accumulation by Adrenergic Receptors in Hypothalamic and Preoptic Area Slices: Effects of Estradiol

Adrenergic receptor agonists and antagonists were employed to establish (a) which receptor subtypes mediate the cyclic AMP response to norepinephrine in hypothalamic and preoptic area slices from gonadectomized female rats and (b) which receptor subtypes might be modulated by the steroid hormone estradiol. Slice cyclic AMP levels were elevated by the beta receptor agonist isoproterenol, but not by alpha 1 (phenylephrine, methoxamine) or alpha 2 (clonidine) agonists. However, the alpha agonist phenylephrine potentiated the effect of the beta agonist isoproterenol on slice cyclic AMP accumulation. In slices from rats given no hormone treatment, the beta antagonist propranolol inhibited norepinephrine-stimulated cyclic AMP production, while the alpha 1 antagonist prazosin was without effect. In contrast, the cyclic AMP response to norepinephrine in slices from estradiol-treated rats was blocked more effectively by prazosin than by propranolol. Estradiol treatment also attenuated the production of cyclic AMP by the beta agonist isoproterenol. The data suggest (a) that norepinephrine induction of cyclic AMP accumulation in hypothalamic and preoptic area slices is mediated by beta receptors and potentiated by alpha receptor activation and (b) that estradiol depresses beta and increases alpha 1 receptor function in slices from brain regions associated with reproductive physiology.     

Properties of receptor-controlled inositol trisphosphate formation in parotid acinar cells.

Activation of muscarinic receptors in rat parotid cells results in breakdown of polyphosphoinositides liberating inositol phosphates, including inositol trisphosphate. Formation of inositol trisphosphate appears independent of agonist-induced Ca2+ mobilization, since neither formation nor degradation of inositol trisphosphate are appreciably altered in low-calcium media, and elevation of cytosolic Ca2+ with a calcium ionophore does not cause an increase in cellular inositol trisphosphate. Further, activation of substance P receptors and alpha 1-adrenoreceptors, but not beta-adrenoreceptors, increases inositol trisphosphate formation. The dose-response curve for methacholine activation of inositol trisphosphate formation more closely approximates the curve for receptor occupancy than for Ca2+-activated K+ release. These results are all consistent with the suggestion that inositol trisphosphate could function as a second messenger linking receptor occupation to cellular Ca2+ mobilization.     

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.     

Mechanisms of leukotriene E4 partial agonist activity at leukotriene D4 receptors in differentiated U-937 cells

Leukotriene E4 (LTE4) is shown to be a partial agonist of leukotriene D4 (LTD4) in differentiated U-937 cells. The data that support this conclusion are: 1) LTE4 completely displaced [3H]LTD4 from its receptors in U-937 cell membranes. 2) LTE4 induced only 30 +/- 4% of the maximal Ca2+ transient induced by LTD4 in the presence of 1 mM extracellular Ca2+ and 60 +/- 4% of the maximal LTD4 response in the absence of extracellular Ca2+. 3) LTE4 induced only a fraction of the inositol phosphates metabolized by LTD4. Moreover, LTE4 resulted in essentially no production of the inositol 1,4,5-trisphosphate isomer, while LTD4 induced a rapid and substantial transient increase in this isomer. The generation of inositol phosphates by both agonists was unaffected by extracellular Ca2+. 4) The EC50 values for Ca2+ mobilization for LTD4 and LTE4 corresponded with their affinity (Kd values) for the LTD4 receptor. 5) A series of structurally diverse LTD4 receptor antagonists blocked the Ca2+ mobilization responses to LTD4 and LTE4 with identical rank orders of potency. 6) LTE4 acted as an antagonist of LTD4 of potency. 6) LTE4 acted as an antagonist of LTD4 effects when they were coadministered. 7) LTE4 and LTD4 acutely desensitized Ca2+ mobilization to each other. All of the effects of LTE4 are explained by its partial agonist activity at the LTD4 receptor as shown by the following data. 1) Neither LTD4 nor LTE4 had any effect on the agonist activity of fMet-Leu-Phe, LTB4, or platelet-activating factor. 2) None of the above agonists or antagonists to the above receptors affected any of the activities of LTD4 or LTE4. 3) Neither LTD4 nor LTE4 induced desensitization of Ca2+ mobilization to any of the non-LTD4 receptor agonists tested. 4) Under the conditions studied, we have not observed any evidence of multiple subclasses of LTD4 receptors in U-937 cells. LTE4 is a partial agonist of the LTD4 receptor, because it can only couple the LTD4 receptor to a portion of the signaling system available to the receptor when occupied by LTD4. Specifically, LTD4 caused the activation of receptor-operated calcium channels, mobilization of intracellular Ca2+, the activation of phosphatidylinositol-phospholipase C, and the liberation of an additional, as yet undefined, intracellular mediator. To do this, LTD4 receptors couple to at least two and perhaps more guanine nucleotide binding proteins. LTE4 is unable to activate the phosphatidylinositol-phospholipase C but can mimic the other effects of LTD4.(ABSTRACT TRUNCATED AT 400 WORDS)