Gi is involved in ethanol inhibition of L-type calcium channels in undifferentiated but not differentiated PC-12 cells.

The effects of acute exposure to 25 mM ethanol on high voltage-activated, L-type Ca2+ channels in undifferentiated and nerve growth factor-treated pheochromocytoma (PC-12) cells were examined using conventional, whole-cell, patch-clamp techniques. Acute exposure to 25 mM ethanol inhibited macroscopic L-type Ca2+ currents in undifferentiated PC-12 cells significantly more than in nerve growth factor-treated PC-12 cells. Intracellular infusion with guanosine-5'-O-(2-thio)diphosphate or pretreatment with pertussis toxin reduced ethanol inhibition in undifferentiated cells without altering inhibition in nerve growth factor-treated cells, suggesting the involvement of a G protein in ethanol inhibition of Ca2+ channels in undifferentiated cells. Intracellular infusion with an affinity-purified antibody that recognizes the carboxyl termini of alpha i1 and alpha i2 significantly reduced ethanol inhibition in undifferentiated cells, in contrast to the effects of antibodies that recognize the carboxyl termini of alpha oA and alpha oB. None of these antibodies reduced ethanol inhibition in nerve growth factor-treated cells. These results indicate that Gi1 alpha or Gi2 alpha mediates ethanol inhibition of L-type Ca2+ channel currents in undifferentiated but not in nerve growth factor-treated PC-12 cells.

Inhibition of dihydropyridine-sensitive Ca++ channels by ethanol in undifferentiated and nerve growth factor-treated PC12 cells: interaction with the inactivated state.

The effects of acute exposure to ethanol on high-voltage activated calcium (Ca++) channels in undifferentiated (UND) pheochromocytoma (PC12) cells and in PC12 cells treated with nerve growth factor (NGF) were compared using the nystatin perforated-patch voltage-clamp technique. Both UND and NGF-treated cells expressed noninactivating, dihydropyridine-sensitive Ca++ current. Ethanol (10, 25 and 50 mM) reversibly inhibited noninactivating current in both UND and NGF-treated cells. However, the reduction of current was significantly greater in UND cells. NGF-treated cells expressed, in addition to dihydropyridine-sensitive current, omega-conotoxin GVIA-sensitive Ca++ current. The reduced ethanol inhibition in NGF-treated cells was not due to the contribution of these channels to the overall current since conotoxin did not alter the level of ethanol inhibition. In both cell types, ethanol had no significant effect after exposure of the cells to nifedipine, suggesting that these drugs act on the same dihydropyridine-sensitive Ca++ channel type. In both cell types, inhibition was greatest when voltage steps were made from depolarized holding potentials, suggesting that ethanol has a greater effect on the inactivated state than on the resting or activated state. Steady-state inactivation measurements indicated that ethanol caused a significant hyperpolarizing shift in the half-maximal inactivation potential for both cell types, supporting this proposition. The magnitude of this shift was correlated with the magnitude of ethanol inhibition of currents elicited from a holding potential of -40 mV. Thus, one mechanism by which ethanol inhibits Ca++ currents in PC12 cells is via a shift in steady-state inactivation An additional mechanism in UND cells must underlie the difference in ethanol sensitivity of Ca++ channels in the two cell types.

Time course of nerve growth factor modulation of ethanol inhibition of Ca2+ currents in PC12 cells.

The effects of 25 mM ethanol on high voltage-activated (HVA) L-type Ca2+ channels in nerve growth factor (NGF)-treated, dibutyryl cyclic AMP (dbcAMP)-treated and undifferentiated (UND) PC12 cells were examined. Channels in NGF-treated cells became less sensitive to the effects of ethanol. The time course of the NGF-induced reduction in ethanol inhibition occurred in two phases. The first phase of reduced inhibition was observed after 2 h of NGF exposure and was complete after 5 h. The second phase of reduced inhibition developed over a period of 10-96 h and was reversible after removal of NGF. Exposure to dbcAMP for 2 h also caused a transient drop in ethanol inhibition, but the second phase of decreased ethanol sensitivity was absent. Thus, increased levels of cAMP do not appear to be involved in the longer-term effects of NGF on the decrease in ethanol inhibition of Ca2+ channels, whereas the effects observed at 2 h may involve cAMP.

Ethanol inhibition of L-type Ca2+ channels in PC12 cells: role of permeant ions.

The effects of acute ethanol exposure on voltage-activated Ca2+ channels in undifferentiated pheochromocytoma (PC12) cells were investigated using whole-cell patch clamp techniques. Concentrations of ethanol (5, 25 and 50 mM), at or below blood alcohol levels which constitute legal intoxication significantly reduced Ca2+ currents evoked from a holding potential of -30 mV. Ethanol-induced inhibition of current was voltage-dependent in some cases, but this was not consistently observed. Inhibition of currents was reversible and was not due to an osmotic effect. The non-inactivating nature of the current, the inhibition of the current by nifedipine, and the lack of inhibition by omega-conotoxin, indicated that the current was carried through high-voltage activated, L-type Ca2+ channels. Since intracellular Ca2+ levels were highly buffered by exchanged with the contents of the patch pipet, ethanol-induced inhibition of currents in PC12 cells is not likely to involve either a change in driving force due to a change in intracellular Ca2+ levels or potentiation of Ca(2+)-dependent Ca2+ channel inactivation by the influx of Ca2+. The degree of inhibition by 25 mM ethanol was the same when either Ca2+, Ba2+ or Na+ was used as the current-carrying ion. This equivalency suggest that the channel's ion selectivity filter is not a site of action for ethanol.

Electrophysiological studies on calcium channels in naive and ethanol-treated PC12 cells.

In this paper, we describe the effects of both acute and chronic ethanol exposure on voltage-activated calcium channels in undifferentiated pheochromocytoma (PC12) cells. Calcium uptake and calcium mediated release studies have shown that calcium channels in these cells are sensitive to ethanol (Messing et al., 1986, Rabe and Weight, 1988, Koski et al., 1991 and Grant et al. in press). Using whole-cell patch clamp techniques, we show that the electrical properties of the channels are very sensitive to acute ethanol. Calcium currents were significantly inhibited by 25 mM acute ethanol and this effect was reversible. In contrast to the reduction of current seen with acute ethanol exposure, chronic ethanol exposure of the cells resulted in a significant increase in calcium current. Examination of whole-cell calcium currents suggested the development of tolerance to ethanol in PC12 cells. Currents evoked from cells exposed to chronic ethanol were inhibited less by acute ethanol than currents from cells not exposed to chronic ethanol. Collectively, these data demonstrate that calcium channels in PC12 cells are sensitive targets for both acute and chronic ethanol.

Regulation of adenohypophyseal messenger RNAs in female rats by age, hypothyroidism, estradiol and neonatal androgenization.

Hormonal regulation of adenohypophyseal messenger ribonucleic acids (mRNAs) encoding preprotachykinin (PPT), prolactin (PRL) and thyrotropin beta subunit (TSH beta) was examined in juvenile and pubertal female rats. Hypothyroidism, initiated on day 2 (d2) or 22 (d22) of life, increased PPT and TSH beta mRNAs but decreased PRL mRNA 17 days later. Exogenous estradiol given for 3 days reduced PPT mRNA in pubertal (d38) but not juvenile (d18) euthyroid females; conversely, estradiol increased PRL mRNA on d18 but not d38. In hypothyroid females however, estradiol decreased PPT and TSH beta mRNAs at both ages and increased PRL mRNA in pubertal but not juvenile females. Thus, regulation of adenohypophyseal mRNAs by estradiol varies with age and thyroid status. In previous studies, adenohypophyseal tachykinins increased in male, but not female rats at puberty. This sex difference was not reproduced here by neonatal androgenization of females, suggesting that it is not mediated by hypothalamic sexual differentiation. However, PRL mRNA increased in androgenized females; this increase was prevented by ovariectomy, suggesting its medication by estradiol.

Ligand binding and G protein coupling of muscarinic receptors in airway smooth muscle.

Ligand binding properties of muscarinic receptors were examined in membranes and isolated cells prepared from bovine trachea. The binding of the muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) to both membranes and isolated cells was saturable, reversible, and of high affinity [dissociation constant (KD) = 100-200 pM]. The binding constants of three selective antagonists, pirenzepine, AF-DX 116, and 4-DAMP, were examined, and the results indicate that the smooth muscle cells contain at least two receptor subtypes. The majority of receptors exhibit binding constants for these selective antagonists similar to those of the M2-subtype. AF-DX 116 binding curves indicated the presence of a small population of receptors with binding constants similar to those reported for the M3-subtype. These findings suggest that the smooth muscle cells may contain both M2- and M3-receptors and are in agreement with evidence of the presence of mRNAs coding for these two subtypes in tracheal extracts (A. Maeda, T. Kubo, M. Mishina, and S. Numa. FEBS Lett. 239: 339-342, 1988). [3H]QNB displacement curves of the muscarinic agonist oxotremorine were best described as a sum of binding to high- and low-affinity sites with KD values of 3.8 nM and 2.2 microM. Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) shifted the high-affinity sites to low affinity, suggesting that the high-affinity sites may represent receptors coupled to G proteins. Pertussis toxin catalyzed the ADP ribosylation of a 40- to 41-kDa protein band present in the membranes but had no significant effect on high-affinity agonist binding, suggesting that most of the receptors are coupled to G proteins in a toxin-insensitive manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Substance P and neurokinin A in the cat carotid body: localization, exogenous effects and changes in content in response to arterial pO2.

In the present work we studied the occurrence of substance P (SP) and neurokinin A (NKA) in the carotid bodies of cats by means of immunocytochemistry and radioimmunoassay (RIA). We also compared the exogenous effects of SP and NKA on carotid body sensory discharge. SP- and NKA-like immunoreactivities (SP-LI; NKA-LI) were seen in many glomus cells and in a sparse plexus of fine fibers. The SP-LI containing glomus cells and fibers also exhibited NKA-LI, suggesting that both these tachykinins coexist in the carotid body. Chemoreceptor discharge increased both by SP and NKA in a dose-dependent manner. The peak excitation produced by SP and NKA was the same when the effects were compared on an equimolar basis. The tachykinin content of the carotid bodies varied with changes in arterial pO2. During normoxia, SP and NKA levels were 57 +/- 8 and 85 +/- 14 fmol/mg, respectively. When the animals were exposed for 1 h to 100% O2, SP content was unchanged (51 +/- 4 fmol/mg), whereas NKA levels were significantly lower than during normoxia (29 +/- 3 fmol/mg, P less than 0.01). Following 1 h of hypoxia, SP content of the carotid body was 146 +/- 20 fmol/mg, a value higher than that obtained during normoxia and hyperoxia. NKA levels, on the other hand, were not significantly different from normoxic values. These results indicate that the cat carotid body (1) contains both SP and NKA, (2) both peptides augment neural discharge of the carotid body and (3) their levels in the carotid body are substantially altered by arterial oxygen, the natural stimulus to the chemoreceptors.

Thyroid hormone status regulates preprotachykinin-A gene expression in male rat anterior pituitary.

Substance P (SP) and substance K (SK) are mammalian tachykinin peptides derived from a single preprotachykinin-A (PPT-A) gene and are widely but selectively distributed in neural and endocrine tissues. SP is present in the rat anterior pituitary, and its content there varies with the thyroid status of the animal. The present studies were undertaken to determine whether the PPT-A gene is expressed in the anterior pituitary and if so, whether PPT-A messenger RNA (mRNA) abundance is regulated by thyroid hormone status. Male rats were surgically or chemically thyroidectomized or made hyperthyroid by thyroid hormone (T3) injection. Total RNA was isolated from individual anterior pituitaries, and PPT-A mRNA abundance was determined by dot blot procedures. In parallel groups of rats, anterior pituitaries were extracted for measurement of SP and SK by specific RIAs. Surgical or chemical thyroidectomy increased PPT-A mRNA abundance 4 to 6-fold and increased both SP and SK content in the anterior pituitary. Administration of T3 to thyroidectomized rats reversed the increase in both PPT-A mRNA abundance and SP and SK content in the adenohypophysis. T3 administration to euthyroid rats also decreased PPT-A mRNA abundance and SP and SK content in the anterior pituitary. The coordinate presence of PPT-A mRNA with SP and SK in the anterior pituitary strongly suggests that these peptides are synthesized within this gland.

Protection of opiate receptors in NG108-15 against modification by N-ethylmaleimide.

Two different -SH groups associated with the opiate receptors of the mouse neuroblastoma X rat glioma hybrid NG108-15 have been identified. Modification of these by N-ethylmaleimide (NEM) (presumed to be via alkylation) or by para-chloromercuribenzoic acid (presumed to be via formation of mercury adducts) decreases the binding of both opiate agonists and antagonists to these receptors. Agonist binding is more sensitive than antagonist binding to modification by NEM. Losses in antagonist binding are accounted for totally by decreases in the number of binding sites; there are no corresponding losses in antagonist affinity. Losses of antagonist binding exhibit a pseudo-first order rate constant; the modification of only one such group completely destroys the binding site. Both agonists and antagonists protect against modification of this group by NEM. Sodium and lithium, but not GTP, also protect this group, indicating that the action of these monovalent cations is directly on the receptor moiety. Losses in agonist binding stem not only from decreases in receptor number but also from selective losses in affinity. This -SH group appears to be different from the one at the binding site as sodium, GTP, and antagonist ligands do not protect against losses in agonist affinity. Agonist high affinity also is lost in a pseudo-first order fashion indicating that an alteration of only one -SH group per receptor complex is sufficient to produce this effect. The possible roles of two sulfhydryls in opiate receptor function are discussed.

Hyperpolarization of neuroblastoma-glioma hybrid NG108-15 by vanadium ions.

Vanadate hyperpolarizes mouse neuroblastoma-glioma hybrid NG108-15 by 20-30 mV. These changes in membrane potential (delta psi) are observed by monitoring the equilibrium distribution (intracellular/extracellular) of the lipophilic cation [3H]tetraphenylphosphonium (TTP+) and by directly measuring delta psi with intracellular microelectrodes. In physiological media (i.e., 135 mM NaCl/5 mM KCl), the half-maximal effective concentrations of sodium orthovanadate (Na3VO4) and sodium metavanadate (NaVO3) are 35 and 160 microM, respectively. The maximal effects for both these ions are quantitatively indistinguishable. The hyperpolarizing responses to vanadate occur without any observable lag, have t1/2 less than or equal to 30 sec, and are always accompanied by simultaneous decreases in membrane resistance. Neither ouabain nor media containing high K (i.e., 120 mM) devoid of Na and K (isotonicity maintained by choline) prevent the change in delta psi induced by vanadate. Therefore, vanadate produces a unique hyperpolarization which does not depend upon Na, K, or the Na/K pump. Furthermore, the accompanying decreases in membrane resistance indicate that vanadate must increase the permeability of the membrane to some ion. Our data are consistent with it being an anion, such as chloride or vanadate itself. Finally, vanadate hyperpolarizes many different types of cultured cells, only some of which are of neuronal origin. This indicates that a hyperpolarization of delta psi must be considered in any assessment of the physiological actions of the vanadates.

Catecholamine-induced desensitization in turkey erythrocytes: cAMP mediated impairment of high affinity agonist binding without alteration in receptor number.

Desensitization of turkey erythrocyte adenylate cyclase by exposure of these cells to the beta-adrenergic agonist isoproterenol leads to a decrease in subsequent adenylate cyclase stimulation by isoproterenol, F-, or Gpp(NH)p without any apparent loss or down regulation of receptors (B.B. Hoffman et al. J. Cyclic Nucl. Res. 5: 363-366, 1979). We now report that the desensitization is associated with a functional "uncoupling" of the beta-adrenergic receptor. This is evidenced by an impaired ability of receptors to form a high affinity, guanine nucleotide sensitive complex with agonist as assessed by computer analysis of radioligand binding data. The changes in adenylate cyclase responsiveness as well as the alterations in receptor affinity for agonists are reproduced by incubation of turkey erythrocytes with the cAMP analog 8-Bromo-adenosine 3':5'- cyclic monophosphate. These findings suggest that one possible mechanism for the development of desensitization in adenylate cyclase systems may be a cAMP mediated alteration of a component(s) of the beta-adrenergic receptor-adenylate cyclase complex which results in impaired receptor-cyclase coupling.

Desensitization of beta-adrenergic stimulated adenylate cyclase in turkey erythrocytes.

Desensitization of catecholamine stimulated adenylate cyclase (AC) activity is demonstrated in membranes derived from turkey erythrocytes pre-treated with isoproterenol. Membranes from desensitized cells had a loss in maximal catecholamine stimulated adenylate cyclase activity of 104 +/- 13 (pmols/mg protein/10', p less than .001) compared with controls. When adenylate cyclase was maximally stimulated with NaF or Gpp(NH)p, the decrements were 84 +/- 19 (p less than .005) and 92 +/- 32 (p less than .05) pmol/mg protein/10' respectively. There was no change in beta-adrenergic receptor number in membranes derived from treated cells. While the molecular mechanism accounting for the desensitization is uncertain, the data is consistent with the hypothesis that there is a lesion distal to the beta-adrenergic receptor, possibly involving the nucleotide site or the catalytic subunit of adenylate cyclase, causing the desensitization in the isoproterenol treated cells.