The relationship between alpha 1-adrenergic receptor occupation and the mobilization of intracellular calcium

We have simultaneously quantitated alpha 1-adrenergic receptor occupation and agonist-elicited Ca2+ mobilization monitored as unidirectional 45Ca2+ efflux from intact BC3H-1 muscle cells in order to examine the relationship between the number of surface receptors occupied and the functional response. [3H]Prazosin has been used to measure receptor number as well as the binding kinetics with surface receptors, and the observed equilibrium and kinetic constants are in close accord with values obtained previously in cellular homogenates. Since alpha 1-agonist-elicited 45Ca2+ efflux can be monitored over intervals of 3 min or less and prazosin dissociation from its receptor has a t 1/2 of 44 min, prazosin can be employed to produce a pseudoirreversible inactivation of receptors. A comparison of the remaining receptors and residual response reveals an inverse linear relationship between receptors inactivated by prazosin and 45Ca2+ efflux. A similar result is obtained following fractional receptor inactivation with the irreversible alkylating agent phenoxybenzamine. Parameters of receptor occupation and response also correlate well for the agonist phenylephrine and for the competitive antagonist phentolamine. The unitary relationship between sites available for occupation and response indicates that the alpha 1 receptor does not function as an oligomer where fewer bound antagonist molecules are required to block the receptor than sites of agonist occupation necessary for activation. Moreover, substantial evidence has accrued in intact smooth muscle for a receptor reserve or nonlinear coupling between alpha 1 receptor occupation and contraction in smooth muscle. Our findings demonstrate that such behavior does not exist for alpha 1 receptor-elicited mobilization of Ca2+ in the BC3H-1 muscle cell.     

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.     

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.     

Dopamine inhibits calcium flux in the 7315a prolactin-secreting pituitary tumour

Cells of the 7315a prolactin-secreting tumour express biochemically normal cell-surface receptors for dopamine. However, dopamine inhibits prolactin release from these cells only when the basal rate of prolactin release is augmented by increasing the intracellular and/or extracellular calcium concentration of the tumour cells. This suggests that dopaminergic modulation of calcium ion flux could have a central physiological role in these neoplastic cells. In 7315a cells we examined the ability of dopamine to regulate 45Ca2+ influx and fractional 45Ca2+ efflux under conditions of enhanced calcium flux using the calcium channel activator, maitotoxin. It was observed that unidirectional calcium influx stimulated by maitotoxin was significantly inhibited by dopamine. Maitotoxin stimulated fractional efflux and prolactin release from the tumour cells and dopamine simultaneously inhibited both processes by a haloperidol-reversible mechanism. Therefore, in 7315a cells dopamine receptor activation is coupled to inhibition of calcium flux as at least one component in the regulation of prolactin release. These cells may provide further opportunity to study intracellular signalling mechanisms that are modulated by dopamine receptor activity.     

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.     

IgE receptor-activated calcium permeability pathway in rat basophilic leukemia cells: measurement of the unidirectional influx of calcium using quin2-buffered cells

The intracellular calcium indicator and buffer quin2 has been used to generate a large calcium buffering capacity in the cytoplasm of rat basophilic leukemia cells. Above 3 mM intracellular quin2, there is no further increase in the initial rate of antigen-induced 45Ca uptake, suggesting that 45Ca buffering by quin2 is now sufficient to prevent the immediate efflux of 45Ca from the cells. Thus, the initial rate of 45Ca uptake should reflect the true unidirectional influx of calcium that occurs when immunoglobulin E (IgE) receptors are aggregated by antigen. The antigen-induced calcium permeability pathway appears to be saturable, with a Km of about 0.7 mM and a Vmax of 0.9 nmol of calcium (10(6) cells)-1 min-1. Although net 45Ca uptake reaches a plateau a few minutes after antigen stimulation, the increase in plasma membrane permeability is maintained for at least an hour, provided that receptors for IgE remain aggregated. The initial rate of 45Ca influx correlates well with the subsequent secretion of [3H]serotonin in response to different concentrations of antigen. Both 45Ca uptake and [3H]serotonin secretion are maximal when only 10% of the receptors are occupied with antigen-specific IgE. Thus, 45Ca influx correlates more closely with secretion than with the number of IgE receptors aggregated by antigen.     

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.     

Comparison of the inhibition of insulin release by activation of adenosine and alpha 2-adrenergic receptors in rat beta-cells.

Rat islets were used to compare the mechanisms whereby adenosine and adrenaline inhibit insulin release. Adenosine (1 microM-2.5 mM) and its analogue N6(-)-phenylisopropyladenosine (L-PIA) (1 nM-10 microM) caused a concentration-dependent but incomplete (45-60%) inhibition of glucose-stimulated release. L-PIA was more potent than D-PIA [the N6(+) analogue], but much less than adrenaline, which caused nearly complete inhibition (85% at 0.1 microM). 8-Phenyltheophylline prevented the inhibitory effect of L-PIA and 50 microM-adenosine, but not that of 500 microM-adenosine or of adrenaline. In contrast, yohimbine selectively prevented the inhibition by adrenaline. Adenosine and L-PIA thus appear to exert their effects by activating membrane A1 receptors, whereas adrenaline acts on alpha 2-adrenergic receptors. Adenosine, L-PIA and adrenaline slightly inhibited 45Ca2+ efflux, 86Rb+ efflux and 45Ca2+ influx in glucose-stimulated islets. The inhibition of insulin release by adenosine or L-PIA was totally prevented by dibutyryl cyclic AMP, but was only attenuated when adenylate cyclase was activated by forskolin or when protein kinase C was stimulated by a phorbol ester. Adrenaline, on the other hand, inhibited release under these conditions. It is concluded that inhibition of adenylate cyclase, rather than direct changes in membrane K+ and Ca2+ permeabilities, underlies the inhibition of insulin release induced by activation of A1-receptors. The more complete inhibition mediated by alpha 2-adrenergic receptors appears to result from a second mechanism not triggered by adenosine.     

Magnesium effects on activation of skinned fibers from striated muscle

The intracellular Ca movements that control contraction and relaxation of striated muscle are regulated by the membrane potential and influenced by Mg2+. In skinned fibers, the internal composition can be manipulated directly by Ca movements estimated from isometric force transients, net changes in sarcoplasmic reticulum (SR) Ca, and 45Ca flux between fiber and bath. Stimulated Ca release, unlike unstimulated 45Ca efflux at low external [Ca2+], is highly [Mg2+]-sensitive at 20 C. Force and tracer measurements indicate three major sites of Mg2+-Ca2+ interaction in situ: Mg2+ can stimulate the SR active Ca transport system, inhibit a Ca2+-dependent Ca efflux pathway of SR, and shift the force-[Ca2+] relation, presumably by reducing Ca2+ binding to myofilament regulatory sites. These mechanisms constrain the resting Ca flux and are adaptive during relaxation. However, analysis of CI-stimulated 45Ca release and reaccumulation suggests that the depolarization process may inhibit Mg2+-dependent Ca influx, the membrane potential controlling both efflux and influx; recent studies on voltage-clamped cut fibers support this hypothesis. The Ca2+ and Mg2+ dependence of caffeine-stimulated 45Ca efflux suggests that Mg2+ inhibition of the Ca2+-dependent efflux pathway is small during rapid Ca2+ efflux. Therefore, both Mg2+ mechanisms, which minimize net release, may be reversed during normal activation.     

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.     

Extracellular calcium ion depletion in frog cardiac ventricular muscle.

The extracellular free [Ca++] in frog ventricular muscle strips was monitored using single-barrel calcium ion-selective microelectrodes. During trains of repetitive stimulation, a heart rate-dependent, sustained fall (depletion) of the extracellular free [Ca++] occurs, which is most likely a consequence of net Ca++ influx into ventricular cells. The magnitude of the [Ca++]0 depletion increases for higher Ringer's solution [Ca++], and is reversibly blocked by manganese ion. Prolonged repetitive field stimulation (20-30 min) activates additional cellular Ca++ efflux, which can balance the additional Ca++ influx caused by stimulation, resulting in abolition of extratrabecular [Ca++]0 depletion in 20-30 min, and hence zero net transmembrane Ca++ flux at steady state. In the poststimulation period of quiescence, cellular Ca++ efflux persists and causes an elevation (accumulation) of the extracellular free [Ca++]. From these [Ca++]0 depletions, quantitative estimates for the net transmembrane Ca++ flux were derived using an analytical solution to the diffusion equation. In the highest Ringer's solution [Ca++] used (1 mM) the calculated net increase of the total intracellular calcium per beat was 6.5 +/- 1.4 mumol/l of intracellular space. This corresponds to an average net transmembrane Ca++ influx of 0.81 +/- 0.17 pmol/cm2/s during the 800-ms action potential. In lower bath [Ca++] the net transmembrane [Ca++] flux was proportionately reduced.     

Characterization of benextramine as an irreversible alpha-adrenergic blocker and as a blocker of potassium-activated calcium channels

An irreversible alpha-adrenergic blocker, benextramine [N,N'-bis(o-methoxybenzylamine-n-hexyl)-cysteamine] was used as a probe to study the possible interrelationship between alpha-adrenoceptors and the K+-activated Ca2+-channels. Benextramine, a tetraamine disulfide, acts irreversibly both on the alpha 1-adrenoceptor (t 1/2 = 3 min) and the alpha 2-adrenoceptors. These studies were carried out on rat brain synaptosomes, [3H]prazosin and [3H]clonidine binding. Benextramine blocked Ca2+ influx in rat brain synaptosomes under both depolarizing (75 mM KCl) and normal conditions (5 mM KCl). Its action at the channel is reversible with IC50 = 10 +/- 5 microM of the net Ca2+ influx. This makes benextramine a most potent Ca2+ blocker compared to verapamil or nicardipine (IC50 = 200 microM and 170 microM, respectively). Pretreatment of rat brain slices with benextramine gave a synaptosomal preparation which was devoid of either alpha 1-adrenergic or alpha 2-adrenergic binding capacity due to the irreversible binding of benextramine, but with an undisturbed Ca2+ influx. Thus, these results suggest that the alpha-adrenoceptors and the Ca2+-channels are independent of each other, and that full occupancy of the alpha-receptors does not affect the net calcium flux.     

Receptor regulation of calcium release and calcium permeability in parotid gland cells

The mechanism by which hormones and neurotransmitters regulate fluid secretion in exocrine glands apparently involves the regulation of transmembrane movements of electrolytes, a process for which Ca serves as a second messenger. Analysis of the kinetics of efflux of 86Rb+ (a marker for K+) indicates that the initial phase of the response to secretagogues is mediated through the release of Ca from a cellular pool inaccessible to chelating agents. By investigating the movements of 45Ca under nearly steady-state conditions, we find that this cellular pool can be filled from the extracellular space without a concomitant elevation in ionized intracellular Ca2+. This suggests that the cellular pool is probably associated with the plasma membrane. We have also investigated the possible role of phosphatidic acid in the mechanism by which receptors mobilize Ca2+. Our results suggest that phosphatidic acid, formed on receptor activation, may directly mediate Ca influx into the acinar cell.     

Agonist-stimulated oscillations and cycling of intracellular free calcium in individual cultured muscle cells

Receptor regulation of [Ca2+]i was monitored in individual BC3H-1 muscle cells with intracellularly trapped fura-2 using digital imaging analysis techniques. Activation of alpha 1-adrenergic or H1-histaminergic receptors resulted in multiple bursts, or oscillations, of elevated [Ca2+]i with an average interval frequency of approximately 1.8 min-1. The duration of oscillatory behavior was generally more prolonged in response to phenylephrine than in response to histamine. Additionally, a larger fraction of the cells responded with [Ca2+]i oscillations to phenylephrine (approximately 90%) than to histamine (approximately 60%), although the majority of cells produced oscillations in response to both agonists. In most cells, the receptor-mediated [Ca2+]i oscillations continued for several minutes in the absence of extracellular Ca2+, although the amplitude of the individual peaks gradually decreased. The activation of [Ca2+]i oscillations by H1-receptors was more dependent upon extracellular Ca2+ than those elicited by alpha 1-receptors, reflecting the greater dependency of the histaminergic response on Ca2+ influx. Readdition of Ca2+ to the incubation buffer resulted in the resumption of the [Ca2+]i oscillations. These results indicate that considerable cycling of Ca2+ between the cytoplasm and the endoplasmic reticulum must occur. Receptor-mediated [Ca2+]i oscillations were much more prevalent in subconfluent cells than in confluent cells, possibly due to increased coupling of the cells at higher densities. The cells were capable of responding independently of one another, since sister cells displayed unique temporal responses immediately following cell division. Thus, the linkage of receptor occupancy to [Ca2+]i elevation is a functionally unique property for each individual cell and can be influenced by epigenetic factors.     

Extracellular calcium-dependent regulation of transmembrane calcium fluxes in murine keratinocytes.

Because the level of extracellular Ca2+ is an important stimulus for differentiation of epidermal cells in vitro, we characterized the extracellular Ca(2+)-dependent transmembrane Ca2+ fluxes in BALB/MK mouse keratinocytes. Increasing levels of extracellular Ca2+, ranging from 0.07 to 1.87 mM, stimulated the rate of 45Ca2+ uptake into these cells 10- to 70-fold and doubled the rate of 45Ca2+ efflux. The divalent cations, Ni2+ and Co2+, were able to block the influx of Ca2+, but dihydropyridines and verapamil were not. Furthermore, 10 to 100 microM of the trivalent cation La3+ induced a dose-dependent 2- to 100-fold increase of Ca2+ uptake, independently of the level of extracellular Ca2+. These observations suggest that keratinocytes possess a cell-surface "Ca(2+)-receptor," activation of which stimulates the influx of 45Ca2+ through a type of voltage-independent, receptor-operated Ca2+ channels. Epidermal growth factor induced an accumulation of 45Ca2+ of a much smaller magnitude than elevations of the level of extracellular Ca2+, without a detectable increase of Ca2+ efflux. Thus, the divergent cellular responses of keratinocytes to EGF and extracellular Ca2+ may be due, in part, to the distinct changes in transmembrane Ca2+ fluxes that these two stimuli generate. Treatment of cells with type beta transforming growth factor led to a gradual 6-fold increase of the Ca(2+)-activated rate of Ca2+ uptake over a period of 4 hours, but reduced the Ca2+ efflux by approximately 50% within 10 minutes. Thus, type beta transforming growth factor apparently stimulates Ca2+ influx indirectly, but may control the differentiation of keratinocytes by direct inhibition of Ca2+ efflux pumps.     

The cycling of calcium, sodium, and protons across the inner membrane of cardiac mitochondria.

A method is described that permits simultaneous determination of the net charge transfer associated with Ca2+ transport by the ruthenium-red-sensitive carrier and the ionized internal [Ca2+] in heart mitochondria. The data indicate that this carrier catalyses a charge-uncompensated flux of Ca2+. Full charge compensation for Ca2+ influx is provided by the respiration-dependent efflux of H+. The net efflux of Ca2+ induced by Na+ is analysed in terms of two other carriers, a Na+-Ca2+ antiporter and a Na+-H+ antiporter. Evidence is presented that these two carriers are separate and that the Na+-H+ exchange is the more rapid. The fluxes of Ca2+, Na+ and H+ during the Na+-induced efflux of Ca2+ support a series of events in which the Na+-H+ exchange enables unidirectional Ca2+ fluxes via the uniport and antiport systems to be integrated into a cycle.     

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.     

The surface cyclic AMP receptors, cAR1, cAR2, and cAR3, promote Ca2+ influx in Dictyostelium discoideum by a G alpha 2-independent mechanism.

Activation of surface folate receptors or cyclic AMP (cAMP) receptor (cAR) 1 in Dictyostelium triggers within 5-10 s an influx of extracellular Ca2+ that continues for 20 s. To further characterize the receptor-mediated Ca2+ entry, we analyzed 45Ca2+ uptake in amoebas overexpressing cAR2 or cAR3, cARs present during multicellular development. Both receptors induced a cAMP-dependent Ca2+ uptake that had comparable kinetics, ion selectivity, and inhibitor profiles as folate- and cAR1-mediated Ca2+ uptake. Analysis of mutants indicated that receptor-induced Ca2+ entry does not require G protein alpha subunits G alpha 1, G alpha 2, G alpha 3, G alpha 4, G alpha 7, or G alpha 8. Overexpression of cAR1 or cAR3 in g alpha 2- cells did not restore certain G alpha 2-dependent events, such as aggregation, or cAMP-mediated activation of adenylate and guanylate cyclases, but these strains displayed a cAMP-mediated Ca2+ influx with kinetics comparable to wild-type aggregation-competent cells. These results suggest that a plasma membrane-associated Ca(2+)-influx system may be activated by at least four distinct chemoreceptors during Dictyostelium development and that the response may be independent of G proteins.     

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.     

Concanavalin A binding and Ca2+ fluxes in rat spleen cells

Addition of the mitogenic lectin concanavalin A to rat spleen cells results in a small increase in the steady-state Ca2+ content of the cells. 45Ca2+ fluxes were measured under conditions where artifacts due to Ca2+ binding to concanavalin A could be excluded. Both 45Ca2+ influx into and efflux from these cells are significantly activated by the lectin. If 45Ca2+ is added 30 min after concanavalin A the rate of influx is further enhanced. The increase in 45Ca2+ influx correlates well with binding of concanavalin A to the cells. At low concentrations (optimal mitogenic) of the lectin (1 and 3 micrograms/ml) no significant increase in 45Ca2+ influx occurs but an increase in 45Ca2+ efflux is still observed. The results suggest that concanavalin A binding to the cell surface causes an increase in Ca2+ influx into the cells and that activation of Ca2+ efflux occurs as a response to an increase in the cytosolic Ca2+ activity. Thus, Ca2+ may well play a role in triggering lymphocyte activation.