Inhibitory effect of phloretin on histamine release from isolated rat mast cells

The ability of the flavonoid phloretin to inhibit histamine release from rat mast cells varied considerably with the releasing agent investigated. The response to the combination of the ionophore A23187 and the phorbol ester TPA and to suboptimal concentrations of the ionophore (0.5 M) was potently inhibited (IC₅₀ about 5 M), whereas phloretin was less potent against responses to the ionophore (1 M) IC₅₀ of 17 M), to antigen alone and in combination with TPA (IC₅₀ of 30–50 M), to TPA in the absence of calcium (IC₅₀ of 50 M) and to compound 48/80 in the absence and presence of calcium (IC₅₀ of 60–90 M). The inhibition by phloretin at concentrations above 10M was partly counteracted by glucose (5 mM) indicating effects on oxidative metabolism. The flavonoid quercetin was equally potent in inhibiting histamine release induced by antigen, the ionophore at different concentrations and in combination with TPA (IC₅₀ of 20M). Although not conclusive, the results are consistent with an inhibition of protein kinase C by phloretin at concentrations below 10 M. At higher concentrations unspecific actions become apparent and phloretin therefore seems to be of limited utility as a probe for signal-pathways in cell responses.     

Facilitation by 5-hydroxytryptamine of ATP-activated current in rat pheochromocytoma cells

The effects of 5-hydroxytryptamine (5-HT) on an inward current activated by extracellular ATP were investigated in rat pheochromocytoma PC12 cells. Under whole-cell voltage-clamp conditions 5-HT (10 M) reversibly enhanced the amplitude of the current activated by 30 M ATP. The enhancement may not be due to an increase in the number of functional channels because the current activated by 300 M ATP was not remarkably augmented compared with the current activated by 30 M ATP. The current enhancement by 100 M 5-HT was less obvious than that by 10 M 5-HT. When the current kinetics were compared, activation of the ATP-evoked current was accelerated to the same extent by either 10 or 100 M 5-HT, whereas deactivation was largely more accelerated by 100 M 5-HT. Propranolol (10 M), a 5-HT₁ receptor antagonist, or LY53857 (10 M), a 5-HT₂ receptor antagonist, exerted an agonistic effect: the ATP-activated current was facilitated by these compounds. Metoclopramide (10 M), a 5-HT₃ receptor antagonist, neither facilitated the ATP-activated current, nor blocked the current facilitation by 5-HT. Guanosine 5-O-(2-thiodiphosphate) (GDP[S]) (2 mM), the non-hydrolysable analog of guanosine 5-triphosphate (GTP), or K-252a (2 M), a protein kinase inhibitor, did not affect the facilitation by 5-HT of the ATP-activated current when they were included in the intracellular solution. The ATP-activated current pre-facilitated by 10 M dopamine was not enhanced by 10 M 5-HT. Similarly, the pre-facilitation by 5-HT attenuated the current enhancement by dopamine. The results suggest that 5-HT facilitates the ATP-activated channels through receptors that are not readily classified into conventional subclasses of 5-HT receptors. The reciprocal masking between the current facilitation by 5-HT and that by dopamine, combined with their sensitivities to the compounds involved in the intracellular solution, indicates that the facilitation by 5-HT may share not all, but some, common cellular mechanism with that by dopamine.     

Phenotypic differences between induced and spontaneous 2μ-plasmid-free segregants of Saccharomyces cerevisiae

Summary The maximum specific growth rates (_max) of 2 -plasmid-free ([cir°]) segregants of three haploid and one diploid strain of Saccharomyces cerevisiae have been determined and compared with the _max of their 2 -plasmid-containing ([cir ⁺]) progenitors. Two classes of [cir°] strains have been examined: those induced by transformation with a 2 -based recombinant plasmid according to the method of Dobson et al. (1980) and those isolated as spontaneous [cir°] segregants from glucose-limited continuous cultures. The _max of the spontaneous [cir°] segregants was not found to differ significantly from that of their [cir ⁺] parents. In all cases, however, the induced [cir°] strains had a _max which was significantly less than that of their [cir ⁺] counterparts. This effect was particularly marked in the case of the diploid strain where a 34% reduction in _max was observed. The implications of these results are discussed in terms of the effect of the transformation process on host yeast cells.     

On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation

The Ca²⁺ channel subunits _1C-a and _1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba²⁺ current (I _Ba) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 M cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 M PKA and 1 M okadaic acid. The activity of the _1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 M H 89 and was not increased by superfusion with 5 M forskolin plus 20 M isobutylmethylxanthine (IBMX). The _1C-a·₂·₂/ complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 M H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the _1C-a·₂·₂/ channel with 10 M PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca²⁺ current (I _Ca) of cardiac myocytes increased threefold during internal dialysis with 5 M PKA or 25 M microcystin and during external superfusion with 0.1 M isoproterenol or 5 M forskolin plus 50 M IBMX. These results indicate that the L-type Ca²⁺ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.     

Mechanisms underlying facilitation by dopamine of ATP-activated currents in rat pheochromocytoma cells

Mechanisms underlying facilitation by dopamine of extracellular adenosine 5-triphosphate (ATP)-activated current were investigated in rat pheochromocytoma PC12 cells using the whole-cell voltage-clamp techniques. Dopamine (10 and 100 M) augmented the peak amplitude of an inward current elicited by ATP (3–100 M). The activation time course of the ATP-evoked current was accelerated by dopamine; the presence of 10 M dopamine shifted the dependence of activation rate constants on the concentration of ATP toward a lower concentration range two fold. Dopamine also accelerated the inactivation and the deactivation, which was determined from the current decay upon washout of ATP. Intracellular mediators responsible for the dopamine-induced facilitation was estimated by loading various compounds in patch pipettes. Facilitation was not observed when K-252a (1 M), a protein kinase inhibitor, was included in the intracellular solution. In addition, facilitation was also attenuated by intracellular adenosine 5-O-(thiotriphosphate)tetralithium salt (ATPS (1 mM) or --methylene ATP (1 mM). Inclusion of adenosine 3, 5-cyclic monophosphate sodium salt (cAMP, 100 M), guanosine 3,5-cyclic monophosphate sodium salt (cGMP, 100 M), 12-O-tetradecanoylphorbol-13-acetate (TPA, 1 M) or phorbol-12,13-dibutylate (1 M) in the intracellular solution did not affect the facilitation. Guanonsine 5-O-(thiotriphosphate)tetralithium salt (GTPS, 500 M) or guanosine 5-O(2-thiodiphosphate)-trilithium salt (GDPS, 500 M) did not modify the facilitation either. The results suggest that dopamine augments the ATP-activated inward current by facilitating association of ATP to its binding site, and that the augmentation may be mediated through some protein kinase which is different from cyclic-nucleotide-dependent protein kinases or protein kinase C.     

Efficient selection of μ m − mutants from μm-expressing myeloma cells by treatment with ricin A-conjugated anti-μ antibody

We have developed an efficient system for obtaining myeloma mutants defective intrans-acting factors required for immunoglobulin (Ig) gene expression. The system consists of a myeloma cell line designed for this purpose and an efficient method for selecting mutants from it. The cell line is X63.653 transfected with the gene, whose tailpiece sequence was replaced with the transmembrane sequence of human EGF receptor to hold on the cell surface and whose CH1 sequence was removed to prevent from being retained in the endoplasmic reticulum. It efficiently and stably expressed chains of IgM on the cell surface ( _m ⁺ ) without light chains. To obtain mutants lacking _m ( _m ^– ) from the _m ⁺ cell line by selectively killing _m ⁺ cells, a method with ricin A-conjugated anti- antibody was more reliable than complement lysis mediated by anti- antibody. Applying the system, we obtained a variety of _m ^– mutants.     

Role of the GTP-binding protein Gs in the β-adrenergic modulation of cardiac Ca channels

In the heart, the guanosine 5-triphosphate (GTP)-binding protein G_s is activated by hormone binding to -adrenergic receptors and stimulates the intracellular cyclic adenosine 3,5-monophosphate (cAMP) pathway that leads to phosphorylation of L-type Ca channels by the cAMP-dependent protein kinase A [28]. Additionally, G_s can modulate cardiac Ca channels directly in cell-free systems [57]. In order to examine the question of whether these pathways could be separated functionally and whether they act independently or synergistically on L-type Ca channels in intact cells, the whole-cell Ca current (I _Ca) and the respective current density were measured in guinea-pig ventricular myocytes at 0 mV. The following results were obtained.First, typically, the I _Ca density increased from 12 to 40 A/cm² following application of 1 M isoproterenol (ISP) to myocytes bathed in solutions containing 1.8 mM CaCl₂. However, 1 M ISP enhanced I _Ca only from 9 to 17 A/cm² after inhibition of the protein kinase A by dialysis of 0.5 mM Rp-cAMPS (the Rp-isomer of adenosine 3,5-monophosphorothioate) in the presence of 0.5 mM GTP. Withdrawal of GTP from the dialysate attenuated the effects of ISP on I _Ca. Thus, Rpc-AMPS unmasks a GTP-dependent component of the -adrenergic stimulation of I _Ca, which probably reflects the direct stimulation of Ca channels by G_s under block of cAMP-dependent phosphorylation.Second, in cells under dialysis with 100 or 200 M cAMP, bath application of 20–40 M 3-isobutyl-1-methylxanthine (IBMX) enhanced the I _Ca density to about 41 A/cm² indicating saturation of the cAMP pathway. Under this condition, 1 M ISP was without significant effect on I _Ca. This result may suggests that direct G_s stimulation is rather ineffective on Ca channels after maximal cAMP-dependent phosphorylation. Alternatively, maximal stimulation of the cAMP pathway may also interfere with the activation of the G_s pathway in intact myocytes.Third, simultaneous application of 1 M ISP and 40 M IBMX enhanced I _Ca up to densities of around 75 A/cm² during cell dialysis with 100 M cAMP, an effect much stronger than that exerted by IBMX alone under similar conditions. Since it seems likely that G_s is activated more quickly, than the cAMP pathway during application of the ISP/IBMX mixture, the latter result suggests that a direct effect of G_s may act to prime L-type Ca channels for cAMP-dependent phosphorylation during -adrenergic stimulation of cardiac myocytes.     

Effects of vector cutting on its recombination with the chromosomal immunoglobulin gene in hybridoma cells

We have analyzed the effects of linearizing vector DNA on the frequency and pathway of its recombination with the homologous chromosomal gene. The pSV2neo vector bearing a 4.3-kb fragment encoding the mouse immunoglobulin heavy chain constant (C) region was cut either at sites within the C segment or outside C and then transferred to hybridoma cells bearing a mutant gene. The frequency of recombinant cells producing normal was then measured. For most cut sites, whether in regions of homology or of nonhomology, linearization of the transferred DNA enhanced the recombination frequency between the vector and chromosomal genes. When the vector was either uncut or cut at SacI in the region of homology, G418-resistant m⁺ recombinants were found to have integrated the vector by a single reciprocal homologous crossover; the enzyme site (SacI) used for cutting was present in the recombinants. By contrast, when the vector had been linearized at Pvul or SfiI in the region of nonhomology, vector integration involved nonhomologous crossovers, either between transferred DNA molecules or between transferred and chromosomal DNA, and the vector cut sites were absent in these recombinants. Some recombinants were found to have an unaltered as well as recombinant gene, suggesting that the nonhomologous recombination process might have involved sister chromatids.     

Effects of purinergic stimulation on the Ca current in single frog cardiac cells

Ca current (I _Ca) was measured by whole-cell voltage clamp in single cells isolated from frog ventricle, in which the Na current was inhibited by tetrodotoxin (0.3 M) and K currents were blocked by substituting K with 120 mM intracellular and 20 mM extracellular Cs. The influence of stimulation by ATP (0.1–100 M) was assessed in the presence of propranolol (1 M) or pindolol (0.1 M), prazozin (0.1 M) and atropine (10 M). ATP, in the micromolar range, had two types of effect. Like other P₁-purinoagonists, it antagonized the increase in I _Ca elicited by -adrenostimulation. When added alone, 1 M ATP could increase I _Ca up to twofold. An increase in I _Ca was also observed even after it had been maximally enhanced by intracellularly applied cAMP (50 M). Voltage dependence and kinetics of I _Ca were not affected. These effects were considered to be related to P₂-purinoceptor activation. At higher ATP concentrations the increase in I _Ca was less; at 100 M, ATP reduced I _Ca. The ATP-induced increase in I _Ca was prevented by internal perfusion of the cells with GDP [-S] or neomycin, respectively, to block signal transduction to phospholipase C or its phosphodiesterase activity on the polyphosphoinositides. We conclude that P₂purinoceptor stimulation increases the Ca current in frog ventricular cells by a pathway that might involve phosphoinositide turnover.     

Diameter and flow velocity changes of feline small pulmonary vessels in response to sympathetic nerve stimulation

Using an X-ray television system, we measured directly changes in the internal diameter (ID), flow velocity, and volume flow of the small pulmonary vessels (100–500 m ID) in response to electrical sympathetic nerve stimulation (SNS) in anaesthetized cats before and after adrenergic receptor blockade. Flow velocity was obtained by measuring the distance that the leading edge of the contrast medium moved per 0.1 s in the small arteries. Volume flow was obtained from the product of flow velocity and cross-sectional area calculated from the ID of the small arteries. SNS was accolmplished with 10- to 15-V square-wave pulses of 2-ms duration at 20–30 Hz for 20-s periods. In response to SNS, arterial ID decreased significantly by 8–13% in the 200- to 500-m vessels but not in the 100- to 200-m vessels. In the veins, on the other hand, there was no significant ID decrease in any of the 100- to 500-m vessels. After -receptor blockade (phentolamine, 2 mg/kg i.V.), there were significant ID increases (4–9%) in the 100- to 500-m arteries in response to SNS, the maximum increases being in the 100- to 200-m arteries. After -blockade (propranolol, 2 mg/kg i.V.), the ID decrease due to SNS in the 200- to 500-m arteries was enhanced (24–27%) and, in addition, the 100- to 200-m arteries exhibited a significant ID decrease (18%). Combined and -blockade completely abolished the ID decrease due to SNS. In the veins, on the other hand, no ID change occurred even after - or -blockade. The results indicate that SNS selectively constricts 200- to 500-m arteries. The data suggests that SNS has -mediated vasoconstrictor and -mediated vasodilator effects on the 100- to 500-m arteries and that the ID response pattern to SNS depends chiefly on the balance between -mediated vasoconstriction and -mediated vasodilation. Associated with the ID decrease due to SNS, flow velocity was increased by 21%. However, SNS did not affect volume flow, because the increase in velocity was compensated by the reduction in the cross-sectional area (due to the decreased ID).     

Inhibition of the M current in NG108-15 neuroblastoma × glioma hybrid cells

The M current, I _M, a voltage-dependent non-inactivating K current, was recorded in NG108-15 neuroblastoma × glioma hybrid cells, using the whole-cell mode of the patch-clamp technique. We studied inhibition of the M current by bradykinin, phorbol dibutyrate (PDBu), an activator of protein kinase C (PKC), and methylxanthines. Focal application of 0.1–5 M bradykinin inhibited I _M by about 60%; 5 nM bradykinin inhibited by about 40%. Bath application of 0.1 M and 1 M PDBu diminished I _M to about half of the control value. Staurosporine, a PKC inhibitor, applied for 35–43 min in a concentration of 0.3 M significantly reduced the effect of 1 M PDBu. M current blockage by PDBu could be partly reversed by bath application of H-7 (51–64 M), another PKC inhibitor. These observations suggest that the PDBu effect is really due to activation of PKC. The findings are compatible with the view [Brown DA, Higashida H (1988) J Physiol (Lond) 397:185–207] that the bradykinin effect on I _M is mediated by PKC. However, three further observations suggest that this is only true for part of the bradykinin effect. When the suppression of I _M by 1 M PDBu was fully developed, 0.1 M bradykinin produced a further inhibition of I _M. Down-regulation of PKC by long-term treatment with PDBu reduced the effect of 0.1 M bradykinin significantly but did not abolish it. Staurosporine (0.3 M, applied for 31–46 min) failed to reduce the effect of 5 nM bradykinin significantly. The M current could be reversibly blocked by methylxanthines (caffeine, isobutylmethylxanthine, theophylline) in the millimolar range, probably because of a direct action on the M channels.     

Effects of PAF and PAF antagonists on the shape of venous endothelial cellsin vitro

Three platelet-activating factor (PAF) antagonists were tested for their ability to prevent or reduce PAF-induced shape changes of large vein endothelial cellsin vitro. BN52021 had a significant protective action at concentrations of 1 M and 0.1 M, but at 100 M had a damaging effect of its own. CV3988 (0.1 M and 1 M) and L652, 731 (20 M) did not reduce the responses to PAF, and at higher concentrations (CV3988 10 M and 100 M, L652, 731 100 M) both compounds alone caused significant changes of shape. BN52021 (0.1 M) was also effective against leukotriene (LT) C₄, at 1 M against bradykinin and LTE₄, and at 10 M against LTD₄ and the calcium ionophore A23187. BN52021 (10 M) was ineffective against shape changes induced by histamine, prostaglandin (PG) E₂ and lysophosphatidylcholine (LPC). Neither indomethacin (100 M) nor verapamil (20 M) altered the response to PAF.Using electron spin resonance (ESR) spectrometry it was shown that the damaging effects of LPC and CV3988 may be due partly to their detergent properties. It is suggested that the mechanism by which PAF alters the shape of large vein endothelial cells is primarily receptor mediated.     

Fast K channels are more sensitive to riluzole than slow K channels in myelinated nerve fibre

The effects of 1–500 M riluzole, a novel psychotropic agent, were studied on the nodal K current of isolated nerve fibres of the frog. When added to the external solution, the substance rapidly and reversibly inhibited slow, fast 1 and fast 2 K components of the tail K current. The concentrations of riluzole inducing half maximum reduction of slow, fast 1 and fast 2 K conductances were 413 M, 24 M and 21 M respectively. It is concluded that the substance is about 20 times more effective in blocking fast than slow K channels.     

Activity of trimethoprim/ sulfamethoxazole plus polymyxin B against multiresistantStenotrophomonas maltophilia

The inhibitory activity of eight antibiotics and the inhibitory and bactericidal activities of combinations of trimethoprim/sulfamethoxazole (TMP/SMX) plus three fixed concentrations of polymyxin B (0.01 g/ml, 0.1 g/ml and 0.5 g/ml) against 30 multiresistant strains ofStenotrophomonas maltophilia were tested. Polymyxin B at 0.01 g/ml modified the inhibitory activity of TMP/SMX against only 40% of strains. At 0.1 g/ml and 0.5 g/ml, polymyxin B enhanced the inhibitory activity of TMP/SMX activity against all strains. Polymyxin B enhanced the bactericidal activity of TMP/SMX only at concentrations near the minimum inhibitory concentration of polymyxin B alone.     

Soluble β2-μ-Associated and β2-μ-Free HLA Class I Heavy Chain Serum Levels in Interferon-α Nonresponder Chronic Hepatitis C Patients. Markers of Immune Activation, and Response to Antiviral Retreatment

The serum levels of soluble ₂--associated and ₂--free HLA class I heavy chains were determined in 28 interferon- nonresponder chronic hepatitis C patients retreated with interferon- plus ribavirin and in 70 healthy subjects. The baseline levels of ₂--associated and ₂--free HLA class I heavy chains were significantly higher in patients than in healthy controls(P = 0.001). The levels of ₂--associated HLA class I heavy chains significantly increased in responder patients with respect to nonresponders at the third month of treatment(P = 0.03). At the sixth month of treatment and after 6 months of follow up the levels of ₂--associated HLA class I heavy chains decreased in responder patients and increased in nonresponders. The levels of ₂--free HLA class I heavy chains showed only minor changes during and after treatment. We suggest that the determination of hepatitis C virus RNA levels combined with soluble ₂--associated HLA class I heavy chains, as a marker of immune activation, could identify interferon- non responder chronic hepatitis C patients most likely to respond to a retreatment with interferon- plus ribavirin.     

Superfast tension transients from intact muscle fibres

We have measured tension transients from tetanically stimulated intact single fibres of the M. lumbricalus and the M. tibialis anterior of the frog. The measurements were carried out between 5.5 and 7°C at a sarcomere length of 2.1 and 3.1 m with a dsiplacement servo system capable of performing displacements of 50 m in 40 s. We found a very fast recovery process in the tension transients thus obtained and quantified the speed of the observed recovery process in terms of elasticity and damping.If a ramp-shaped input signal of 150 s duration is applied to the system, the velocity is constant over about 100 s. This property of the system enables us to study the velocity-dependent part of the initial drop in tension in more detail. The first part of the tension transients thus obtained, show three phases: an initial rapid drop in tension, a slower steady decrease in tension and a rapid recovery (30 s).     

Nicotinic effects on excitatory field potentials recorded from the immature CA3 area of rat hippocampal slices

We investigated the nicotinic modulation of the excitatory field potentials recorded from the immature (postnatal day 10–20) hippocampal CA3 area, in the presence of the GABA_A antagonist bicuculline methiodide (BMI, 10 M). Nicotine (50 M) enhanced the evoked field potentials; its effects were also observed in the presence of the GABA_B antagonist 2-hydroxy-saclofen (250 M; added to BMI) and were blocked by pre-perfusion with the nicotinic antagonist hexamethonium (HXM, 50 M). The potentiating effects of nicotine in BMI persisted during prolonged perfusion (more than 20 min), while those in control perfusion medium were transient. The nicotinic antagonists HXM (50 M), methyllycaconitine (MLA, 0.01 M) and dihydro--erythroidine (DHE, 50 M) potentiated CA3-evoked field potentials. Perfusion of HXM in the presence of the anticholinesterase eserine (1 M) or the muscarinic antagonist atropine (1 M) did not alter its effects. None of the nicotinic agents tested changed the frequency of spontaneous BMI-induced epileptiform discharges (nicotine, HXM, MLA, DhE), suggesting that nicotinic receptors do not drive spontaneous epileptiform discharges in this in vitro model. These experiments demonstrate that nicotinic receptors are activated tonically during disinhibition and modulate the activity of excitatory synapses in the immature CA3 hippocampal area. The persistent nicotinic facilitatory effects during disinhibition versus the transient in control conditions indicate that nicotinic modulation depends on environmental conditions and also that nicotinic receptors may be a contributing factor in early-life seizures.     

The sensitivity to Zn2+ discriminates between typical and atypical mast cells

The effects of zinc gluconate have been studied on rat peritoneal mast cells and rat basophilic leukemia cells (RBL 2H3) stimulated by various secretagogues. The IC₅₀'s of zinc gluconate on peritoneal cells were (M)1.6, 1.9, 5.4 and 18 for ionophore A23187, phorbol 12-myristate 13-acetate, substance P and immunoglobulin E-antigen, respectively. Higher concentrations of zinc gluconate were required to inhibit histamine secretion from RBL 2H3 cells, i.e. 12 M (ionophore A23187) and 140 M (immunoglobulin E-antigen). Zinc gluconate (10^–4 to 10^–3 M) also inhibited the IgE-dependent contraction of guinea pig trachea but was unable to affect that induced by exogenous histamine. These results suggest that zinc gluconate acts intracellularly and is selective of typical or connective tissue mast cells.     

Tyrosine protein kinase inhibitors prevent activation of cardiac swelling-induced chloride current

The effect of tyrosine protein kinase inhibitors on the swelling-induced chloride current (I _Cl-swelling of dog atrial myocytes was studied using the whole-cell patch-clamp recording technique. Currents were measured during hyperpolarizing voltage ramps with potassium currents blocked by cesium. Osmolarity was varied using mannitol. Exposure to hypotonic solution (249 mosmol/kg) activated I _Cl-swelling. Hypertonic solution ( 363mosmol/kg) was used to shrink swollen cells and turn off I _Cl-swelling. In studies on the acute effect of tyrosine protein kinase inhibitors each cell was swollen three separate times. Control, treatment, and washout I _Cl-swelling were compared. Genistein (50–80 M) prevented reactivation of I _Cl-swelling without affecting cell size. The effect of genistein partially subsided upon washout. The effect of genistein on I _Cl-swelling was not mimicked by 80 M daidzein, a related compound that does not inhibit tyrosine protein kinases. When intracellular adenosine 5-0-(3-thiotriphosphate (ATP[S]) was used, genistein did not prevent the reactivation of I _Cl-swelling. Intracellular ATP[S] did not result in a persistent activation of I _Cl-swelling when cell size was returned to control. Acute exposure to 1 M herbimycin A or 100 M tyrphostin 51 did not prohibit the activation of I _Cl-swelling. A 24-h exposure to 1 M herbimycin A did inhibit I _Cl-swelling. The results provide important clues regarding the activation mechanism for I _Cl-swelling and suggest that a tyrosine protein phosphorylation may be necessary, but not sufficient, for activation of I _Cl-swelling.     

Intrinsic cholinergic excitation in the rat neostriatum: Nicotinic and muscarinic receptors

Summary An in vitro slice technique was employed to study the receptors involved in intrinsic cholinergic excitation in the rat neostriatum. The locally evoked synaptic potentials were suppressed by antinicotinic agents, mecamylamine (10 M), d-tubocurarine (3 M) or hexamethonium (100 M), but not by the antimuscarinic agent atropine (100 M). If the slices were exposed to an acetylcholinesterase (AChE)-inhibitor (paraoxon 1–20 M, physostigmine 0.1–0.5 M), the synaptic potentials were potentiated. The amplitude of the orthodromic population spike increased, and it was further facilitated when the stimulus frequencies were raised from 1–3 Hz to 10–30 Hz. The frequency facilitation following exposure to an AChE-inhibitor was blocked by atropine (1–100 M). Intracellular recording indicated that a slow depolarizing potential caused the frequency potentiation of the orthodromic discharges. Apparently rat neostriatum is similar to cholinergic systems in sympathetic ganglia and spinal Renshaw cells, in that nicotinic receptors mediate fast excitation and muscarinic receptors mediate slow excitation.