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.     

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.     

The effects of indomethacin and verapamil on the shape changes of vascular endothelial cells resulting from exposure to various inflammatory agents

Histamine (300 M), bradykinin (2 M), prostaglandin E₂ (PGE₂) (30 M), or the leukotrienes (LT) C₄ and E₄ (1 M) but not D₄ (1 M) appliedin vitro have been shown to change the shape of endothelial cells lining the guinea pig isolated thoracic inferior vena cava. All caused the formation of inter-endothelial cell gaps. Pre-treatment with either indomethacin (100 M) or verapamil (20 M) reduced the effects of these compounds. It is suggested that indomethacin and verapamil act by reducing the amount of intracellular calcium available for the shortening of contractile protein filaments within endothelial cells.     

Gastric and cardiovascular effects of histamine in the dog

The H-2 receptor stimulation of gastric acid secretion and of heart rate by histamine, a mixed H-1–H-2 agonist, given in 45-min successive step doses 2–150 g base/kg.h and 4(Me)histamine, (4(Me)H), 1.4 to 210 g base/kg.h, a specific H-2 agonist, in five conscious gastric fistula dogs showed no difference between the agonists in ED₅₀s or in maximal responses. The ED₅₀ for acid was lower (15–16 g/kg.h) than for cardiac chronotropism (25 to 27 g/kg.h). Both effects were competitively antagonized by the H-2 antagonist cimetidine, 0.5 to 1 mg/kg.h. Background infusions of diphenhydramine, 2 mg/kg.h, raised the maximum heart rate increase caused by histamine from +108 to +149 beats/min (p<0.05) but not that of 4(Me)H; acid outputs were not augmented. Diphenhydramine 2 mg/kg.h alone caused no significant change in heart rate or blood pressure. Histamine reduced systolic blood pressure (SBP) by 48 mmHg and with diphenhydramine background by 61 mmHg (p<0.05). 4(Me)H at a top dose of 210 g base/kg.h reduced SBP by 81 mmHg (p<0.05). To test whether the effects of added diphenhydramine could be interpreted as an H-1 receptor effect of histamine, the H-1 agonist 2-pyridylethylamine (PEA) was given alone (2–150 g/kg.h in 45-min steps) or as a 100 g/kg bolus during the infusion of 4(Me)H 50 g/kg.h. There was no effect of PEA on gastric acid, heart rate or blood pressure. Even though diphenhydramine augments the effect of histamine on heart rate and blood pressure, the lack of PEA effect would indicate that there is not a direct H-1 receptor mediated effect on gastric acid, heart rate or systolic blood pressure in the conscious dog. This contrasts with published data obtained in anesthetized animals. After a single dose of 4(Me)H, 50 g/kg.h had been infused i.v. for 45 min, acid output had reached 70% of the maximum seen at 90 min, heart rate had increased by 65% of its maximum response but SBP had not yet changed. Cimetidine blocked 4(Me)H-induced hypotension completely but blocked the heart rate increase only partially and competitively. We conclude that in the intact animal there is evidence for a direct H-2 mediated stimulation of heart rate in the conscious dog with kinetics similar to the H-2 stimulation of gastric acid secretion.Supported by Research Grant No. AM09260 from the National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health and by the Medical Research Service of the Veterans Administration.     

In vitro effect of cetirizine on PGE2 release by rat peritoneal macrophages and human monocytes

Cetirizine was first described as a specific anti-H₁ molecule displaying potent antiallergic activity. It was later found that its pharmacological properties extended to cellular actions as on eosinophil recruitment at inflammatory sites in allergic patients. Monocytes and macrophages participate in allergic mechanisms, particularly through high affinity H₁ and H₂ membrane receptors and generation of pro- and anti-inflammatory agents; among them histamine-induced factors, IL-1 and prostanoids are of importance. The aim of this work was to investigate the effect exerted by various concentrations of cetirizine (0.1–10 g/ml) appliedin vitro to human monocytes and peritoneal rat macrophages cultured for 24 h. Peritoneal macrophages were collected either from normal or experimentally inflamed rats. Human monocytes, isolated from peripheral blood, were studied either in a resting state or after stimulation by LPS fromEscherichia coli (1 and 10 g/ml). Cetirizine (10 g/ml) significantly enhanced IL-1 release by human monocytes stimulated by a weak LPS concentration (1 g/ml) but could not modify the maximal increase of IL-1 release induced by 10 g/ml of LPS. It did not exert any effect on resting cells. Cetirizine (0.1–10 g/ml) enhanced PGE₂ release by resting human monocytes. Concentrations of 1 and 10 g/ml enhanced PGE₂ release by LPS-stimulated monocytes, and by healthy and inflamed rat macrophages. This effect was concentration-dependent. Our findings point to an anti-inflammatory action of cetirizine via PGE₂ release and histamine H₂ interactions. Cetirizine did not directly modify IL-1 generation by resting monocytes but the IL-1 production observed after LPS stimulation could promote the mechanisms by which PGE₂ is released.     

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.     

Electrophysiological characterization of histamine receptor subtypes in sheep cardiac Purkinje fibers

The histamine-receptor-subtype-mediated effects on action potentials of electrically driven and spontaneously active isolated sheep cardiac Purkinje fibers were investigated using H₁-and H₂-selective agonists and antagonists.In electrically stimulated Purkinje fibers, histamine (3 mol/l) increased the action potential plateau height, decreased the action potential duration measured at a repolarization level of –60 mV and enhanced the pacemaker activity. These effects were abolished by the H₂-selective antagonist cimetidine (30 mol/l), but were not impaired by the H₁-selective antagonist dimetindene (0.3 mol/l).In spontaneously active Purkinje fibers, histamine (10 mol/l) increased the spontaneous rate by 24%, the slope of diastolic depolarization by 45% and shortened the duration of the diastole by 32% of the respective control measurements. These effects were blocked by 30 mol/l cimetidine, but remained unchanged in the presence of 0.3 mol/l dimetindene.Concentration-response curves of histamine were shifted to the right by approximately 2 logarithmic units in the presence of 30 mol/l cimetidine, but were not influenced in the presence of 0.3 mol/l dimetindene. The H₂-selective agonist impromidine (0.001–0.3 mol/l) had similar actions as histamine on spontaneously active Purkinje fibers, while the H₁-selective agonist 2-(2-pyridyl-)ethylamine was ineffective. It is concluded that the pronounced stimulatory action of histamine on spontaneous activity in sheep cardiac Purkinje fibers is exclusively mediated by H₂ receptors.Dedicated to Prof. Dr. E. Mutschler on the occasion of his 60th birthday.Supported by Ministerium für Wissenschaft und Forschung, Nordrhein-Westfalen, Projekt-Nr. 40008786.     

Evidence for Na+/Ca2+ exchange in isolated smooth muscle cells: a fura-2 study

Isolated smooth muscle cells (SMC) from guinea pig taenia coli were employed. Suspension of cells were externally loaded in saline with the fluorescent calcium indicators quin-2/AM or fura-2/AM at 20–40 M or 4 M respectively, resulting in an estimated intracellular concentration of 100–200 M for quin-2 or 10–20 M fura-2 (free acid). On addition of 100 M carbachol or high K _o ⁺ (80 mM) depolarization, fura-2 loaded cells contracted (104±47 m,n=121 rest: 39±13 m,n=59 contracted) identically to control (103±35 m,n=232 rest: 39±16 m,n=89 contracted) cells, whereas quin-2 loaded cells were unresponsive to these protocols and there was no significant length change. The Ca _i ²⁺ of fura-2 loaded cells was 100±18 nM (mean±SD,n=15) and was not significantly different from quin-2 loaded cells 107±26 nM (n=13). Treatment of fura-2 loaded cells with 100 M ouabain saline for 10–60 min progressively elevated the Ca _i ²⁺ to a mean of 266±83 nM (n=15). Reduction of Na _p ⁺ (96% Li⁺ replaced) significantly increased Ca _i ²⁺ to 317±77 nM (n=8). After pretreatment with ouabain (100 M), Na _o ⁺ replacement (Li⁺) increased Ca _i ²⁺ at a significantly faster rate [3.6 nM min^–1 (control) cf. 19.8 nM min^–1 (ouabain)].     

A dual effect of cardiac glycosides on Ca current in single cells of frog heart

The effects of cardiac glycosides (1 M ouabain, 50 M dihydrooubain, 1 M strophanthidin) on Ca current (I_Ca) were investigated on Csloaded single frog ventricular cells using the wholecell patch-clamp technique (9). Cardiac glycosides exert both inhibitory and stimulatory effects on I_Ca in 20 Cs Ringer solution, but have only a stimulatory effect in O Cs, when the Na,K pump is blocked. The inhibitory response seems related to the inhibition of the Na,K pump by glycosides. The stimulatory effect on I_Ca may contribute to the positive inotropic effect of cardiac glycosides.     

Effect of steroids on γ-aminobutyrate-induced currents in cultured rat astrocytes

Cultured astrocytes from rat cortex respond to the inhibitory neurotransmitter -aminobutyric acid (GABA) by the activation of Cl^– channels [Bormann J, Kettenmann H (1988) Proc Natl Acad Sci USA 85:9336–9340]. The glial response shares many pharmacological properties with those mediated by neuronal GABA_A receptors, but differs in its sensitivity to inverse benzodiazepine agonists [Backus KH, Kettenmann H, Schachner M (1988) Glia 1:132–140]. To compare glial GABA receptors further with their neuronal counterparts, we analysed the effect of steroids, which have recently been shown to modulate neuronal GABA_A-receptor-mediated responses, on GABA-induced currents in astrocytes. The agonist allotetrahydrodeoxycorticosterone (THDOC) at concentrations of 100 nM and 1 M enhanced GABA-evoked (with 10 M GABA) currents up to 115% and 162.4% of controls respectively. The antagonist dehydroisoandrosterone 3-sulphate (DHEAS) at concentrations of 1 M, 10 M and 100 M depressed GABA-evoked (10 M) currents to 72%, 42.8% and 21.4% of controls respectively. The steroids were less effective at higher GABA concentrations. 100 M DHEAS directly elicited a membrane current, while THDOC (1 M) did not exert any direct response. This study demonstrates that steroids modulate GABA-evoked currents and thus may interfere with any of the functions of glial GABA receptors that are at present under discussion.     

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.     

Different inhibitory effect of etomidate and ketoconazole on the human adrenal steroid biosynthesis

Summary The narcotic agent etomidate and the antimycotic drug ketoconazole are known to block steroid biosynthesis in man. To study the different effects of these imidazole derivatives on human adrenal steroid biosynthesis we incubated slices of human adrenal glands with 3H-labeled precursors and increasing concentrations of etomidate or ketoconazole (0-2000 M). After extraction the labeled metabolites were separated by thin-layer chromatography and quantified by scintillation counting. Etomidate inhibited most potently 11-hydroxylase activity by suppressing the formation of corticosterone from 11-deoxycorticosterone to 1 % of control [50% inhibitory concentration (IC₅₀) 0.03 M] while ketoconazole suppressed 11-hy-droxylase to only 39% of control activity (IC₅₀ 15 M). Ketoconazole however, most potently blocked the conversion of 17-hydroxy-proges-terone to androstenedione by C17,20-desmolase to about 15% of control activity (IC₅₀ 1 M) while etomidate showed a much weaker effect on this enzyme with a suppression to 50% of C17,20-desmolase control activity at a concentration of 380 M. Both imidazole drugs showed a similar strong inhibitory effect on the activity of 17-hy-droxylase (IC₅₀ 6-18 M) and 16-hydroxylase (IC₅₀ 4–8 M) and did not affect 21-hydroxylase. These in vitro data indicate a predominant inhibitory effect of etomidate on corticosteroid biosynthesis by relative selective inhibition of 11-hydroxylase and of ketoconazole on the adrenal androgen biosynthesis by a predominant inhibition of C17,20-desmolase. This differential inhibitory effect of etomidate and ketoconazole on human steroid biosynthesis may be of clinical importance for a possible therapeutic use of these imidazole derivatives in endocrine disorders.Abbreviations IC₅₀ 50% inhibitory concentration Dedicated to Prof. Dr. G. Paumgartner on the occasion of his 60th birthday     

Bile acids increase cellular free calcium in cultured kidney cells (LLC-PK1)

Suspensions of LLC-PK₁ cells were used to determine the effect of bile acids on the cellular homeostasis of morganic ions. It is determined that bile acids alter cellular free calcium (Ca_i) levels in LLC-PK₁ cells. A series of bile acids were compared and found to produce increases in Ca_i in the order: lithocholate sulfate (LCS) > deoxycholate > chenodeoxycholate > lithocholate glucuronide > cholate. LCS (300 M) produces changes in Ca_i (measured using Fura-2) qualitatively similar to those produced by 1 M monomycin, except that only ionomycin is able to release calcium from intracellular stores. The effect on Ca_i is roughly proportional to LCS concentration between 50 and 300 M. The presence of 40 mM Na in the extracellular medium reduces the LCS-induced rise in Ca_i to 20% of that observed in the absence of Na. This effect is specific for Na versus 150 mM extracellular K, Li, or TMA. The effect is not dependent on the Na gradient across the membrane. At concentrations of LCS which induce changes in Ca_i, no significant effect of LCS is observed on either cellular Na or K levels, or intracellular pH.Abbreviations used BCECF 2,7-bis-(2-carboxyethyl)-5,6-carboxy-fluorescein - HEPES 4-(-2-hydroxyethyl)-1-piperazine ethanesulfonate - TMA tetramethylammonium - PCA perchloric acid - LCS lithocholate sulfate - EGTA ethylene glycol-bis-(-aminoethyl ether)N,N-tetra-acetic acid - Ca_i intracellular free calcium - pH intracellular pH     

Synergistic effect of palmitoylcarnitine and the ionophore A23187 on isolated rat mast cells

Dl-palmitoylcarnitine in combination with low concentrations (0.2 M) of the ionophore A23187 induced a pronounced non-cytotoxic histamine release, with maximal response at 10 M palmitoylcarnitine and a lower response at 20 M. The concentration-response curve was shifted to the right when the mast cells were preincubated with palmitoylcarnitine before exposure to the ionophore. Palmitoylcarnitine alone was without effect at concentrations below 20 M but cytotoxic at higher concentrations. The response to the combination of palmitoylcarnitine and the ionophore was highly sensitive to changes in the ionophore concentration. The results indicate that conditions allowing physicochemical interactions between the two drugs led to greatest potency and effectiveness of palmitoylcarnitine. Preincubation with the phorbol ester TPA potentiated the response. The release induced by the combination of palmitoylcarnitine and the ionophore was completely inhibited by low concentrations of the flavonoid phloretin (IC₅₀ of 0.5–2 M) whereas the protein kinase inhibitor H-7 enhanced the response. The synergistic response to palmitoylcarnitine and the ionophore and its affection by phloretin and H-7 resembles previous findings with TPA and the ionophore. Although not conclusive the results indicate that palmitoylcarnitine can stimulate mast cells by activation of protein kinase C.     

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.     

PGE2-induced inhibition of AVP-dependent cAMP accumulation in the OMCD of the rat kidney is cumulative with respect to the effects of α2-adrenergic and A1-adenosine agonists,insensitive to pertussis toxin and dependent on extracellular calcium

The accumulation of cyclic adenosine 3,5-phosphate (cAMP) elicited by antidiuretic hormone (arginine vasopressin, AVP) in the medullary collecting tubule (OMCD) microdissected from the rat kidney is inhibited by different factors: the A₁ agonist of adenosine (-)-N ⁶-(R-phenylisopropyl) adenosine (PIA), an ₂-adrenergic agonist clonidine (CLO), and prostaglandin E₂ (PGE₂). The negative regulation elicited by PGE₂ was further characterized by measuring summation of inhibition with other inhibitors, by testing the effect of pertussis toxin and by studying the part played by extracellular calcium. Inhibitors were used at concentrations inducing maximum effects. The simultaneous addition of 0.3 M PGE₂ with either 0.1 M PIA or 1 M CLO led to an inhibition of the response to AVP (80.0±3.5%, SEM, N=7 and 92.6±0.8%, N=5, respectively) greater than those elicited by each agent alone. In contrast, PIA and CLO added together induced an inhibition similar to that due to CLO alone. The action of PGE₂ in combination with either PIA or CLO corresponded to a partial summation fitting with the values calculated by assuming a cumulative inhibition. Preincubation of OMCD samples with pertussis toxin (100 ng/ml or 1 g/ml) relieved the inhibitory effects of CLO and PIA but did not affect the action of PGE₂. PGE₂-induced inhibition was prevented in a calcium-free medium [0 Ca²⁺+0.1 mM [ethylene-bis (oxyethylenenitrilo)] tetraacetate (EGTA)]: values were 67.0 ±2.1% and 5.8±8.7% (± SEM) in 2 mM Ca²⁺ and 0 Ca²⁺ medium, respectively, N=7. When applied to Fura-2-loaded OMCD, 0.3 M PGE₂ increased intracellular calcium concentration ([Ca²⁺]_i) with a peak phase (in 2 mM or 0 Ca²⁺ medium) followed by a plateau phase (observed only in 2 mM Ca²⁺ medium). It is concluded that: (1) in the rat OMCD, PGE₂, PIA and CLO act on the same AVP-sensitive cell, (2) PGE₂ induces a cumulative inhibition on the cAMP level when combined with other inhibitors by a mechanism insensitive to pertussis toxin, (3) the presence of extracellular calcium is a prerequisite condition to observe PGE₂-induced inhibition, and (4) the inhibition by PGE₂ might be linked to its capacity of increasing [Ca²⁺]_i.     

Ultrastructure and quantitative synaptology of the sacral parasympathetic nucleus

Summary This study examines the anatomical substrate for the spinal micturition reflex. Light microscopy of pyridine silver-stained sections revealed that the sacral parasympathetic nucleus (SPN) exists as a broken column or chain of cell clusters located along the intermediolateral portion of the dorsal horn in sacral segments S₂–S₄. Quantitative analysis of neuropil components in electron micrographs provides data for each type of bouton identified in this nucleus. On the somata of these neurons, boutons containing clear spherical vesicles (S type) comprise 70% of the bouton population. Terminals containing three or more dense core vesicles (GS boutons) account for 26% and boutons containing flattened vesicles (F boutons) comprise 4% of the population. F boutons are more common on large dendrites where they comprise 10% of the total bouton population.The actual population density of each bouton type is most evident when the number of boutons is expressed as boutons per 100 m of membrane length (btn/100 m). S type boutons are the most frequently encountered type. The population density of S boutons is the same on soma and dendrites at 6.66 btn/100 m. F boutons are more numerous on large (> 2 m) dendrites (1.28 btn/100 m) than on small dendrites (0.63 btn/100 m) or on somata (0.36 btn/ 100 m). GS boutons occur more frequently on small dendrites (3.66 btn/100 m) than on somata (2.29 btn/100 m), large dendrites (2.88 btn/100 m) or medium dendrites (2.27 btn/ 100 m). These data suggest that the dense core vesicle-containing boutons are applied primarily to small (<1 m) dendrites and that F boutons are associated mostly with large or proximal dendrites.These results provide a quantitative profile of the synaptic input to the sacral autonomic (parasympathetic) neurons which innervate the urinary bladder and demonstrate specific population differences on various postsynaptic structures in this nucleus.     

β-Adrenoceptor stimulation activates large-conductance Ca2+-activated K+ channels in smooth muscle cells from basilar artery of guinea pig

We studied the effect of isoproterenol on the Ca²⁺-activated K⁺(BK) channel in smooth muscle cells isolated from the basilar artery of the guinea pig. Cells were studied in a whole-cell configuration to allow the clamping of intracellular Ca²⁺ concentration, [Ca²⁺]_i. Macroscopic BK channel currents were recorded during depolarizing test pulses from a holding potential (V _H) of 0 mV, which was used to inactivate the outward rectifier. The outward macroscopic current available from aV _H of 0 mV was highly sensitive to block by external tetraethylammonium·Cl (TEA) and charybdotoxin, and was greatly augmented by increasing [Ca²⁺]_i from 0.01 to 1.0 M. With [Ca²⁺]_i between 0.1 and 1.0 M, 0.4 M isoproterenol increased this current by 58.6±17.1%, whereas with [Ca²⁺]_i at 0.01 M a sixfold smaller increase was observed. With [Ca²⁺]_i0.1 M, 100 M dibutyryl-adenosine 3:5: cyclic monophosphate (cAMP) and 1 M forskolin increased this current by 58.5±24.1% and 59.7±10.3%, respectively. The increase with isoproterenol was blocked by 4.0 M propranolol extracellularly, and by 10 U/ml protein kinase inhibitor intracellularly. Single-channel openings during depolarizing test pulses from aV _H of 0 mV recorded in the whole-cell configuration under the same conditions (outside-outwhole-cell recording) indicated a slope conductance of 260 pS. In conventional outside-out patches, this 260-pS channel was highly sensitive to block by external TEA, and in inside-out patches, its probability of opening was greatly augmented by increasing [Ca²⁺]_i from 0.01 to 1.0 M. Outside-out-whole-cell recordings with [Ca²⁺]_i0.1 M indicated that 100 M dibutyryl-cAMP increased the probability of opening of the 260-pS channel by 152±115%. In inside-out patches, the catalytic subunit of protein kinase A increased the probability of opening, and this effect also depended on [Ca²⁺]_i, with a 35-fold larger effect observed with 0.1–0.5 M Ca²⁺ compared to 0.01 M Ca²⁺. We conclude that the BK channel in cerebrovascular smooth muscle cells can be activated by-adrenoceptor stimulation, that the effect depends strongly on [Ca²⁺]_i, and that the effect is mediated by cAMP-dependent protein kinase A with no important contribution from a direct G-protein or phosphorylation-independent mechanism. Our data indicate that the BK channel may participate in-adrenoceptor-mediated relaxation of cerebral vessels, although the importance of this pathway in obtaining vasorelaxation remains to be determined.     

Nucleotide diphosphates activate the ATP-sensitive potassium channel in mouse skeletal muscle

Patch-clamp techniques were used to study the effects of internal nucleotide diphosphates on the K_ATP channel in mouse skeletal muscle. In inside-out patches, application of GDP (100 M) and ADP (100 M) reversibly increased the channel activity. In the presence of internal Mg²⁺ (1 mM), low concentrations of ADP (<300 M) enhanced channel activity and high concentrations of ADP (>300 M) limited channel opening while GDP activated the channel at all concentrations tested. In the absence of internal Mg²⁺, ADP decreased channel activity at all concentrations tested while GDP had no noticeable effect at submillimolar concentrations and inhibited channel activity at millimolar concentrations. GDP [S] (100 M), which behaved as a weak GDP agonist in the presence of Mg²⁺, stimulated ADP-evoked activation whereas it inhibited GDP-evoked activation. The K⁺ channel opener pinacidil was found to activate the K_ATP channel but only in the presence of internal GDP, ADP and GDP [S]. The results are discussed in terms of the existence of multiple nucleotide binding sites, in charge of the regulation of the K_ATP channel.     

Vasopressin studies in the rat

Summary In previous experiments single i.v. injections of 0.2–10 U ADH were made in alcohol anesthetized rats and the amount of extra water reabsorbed during the antidiuretic phase (urine deficit) was measured. The dose response curve resembled a saturation curve with a fairly linear rise up to 1–1.5 U. When 4–6 U were injected the urine deficit was not appreciably greater while 75% of the ADH injected appeared in the urine during antidiuresis. It was concluded that during a single injection of ADH 1–1.5 U were bound almost instantaneously at receptors of the tubular wall and inactivated during the slow process of water reabsorption, while the excess ADH was excreted in the urine. It was estimated that 1 U ADH was needed for the reabsorption of approximately 5 cm³ water. The time required for this process is short at a high rate of reabsorption and vice versa.In the present investigation the single i.v. injections were repeated with 20 U. The higher dose permitted the separate determination of ADA in 3 consecutive samples of the urine collected during the antidiuretic phase. The result fully confirmed the working hypothesis e.g.:1. The antidiuretic activity (ADA) obtained with 20 U Pitressin was not greater but even (though not significantly) smaller than that obtained previously with 5 U Pitressin or 1 U Tonephin.2. 95±15% of the ADA injected appeared in the urine. This means that the difference between the 20 U injected and the 18.5–19 U appearing in the urine after deduction of the 1–1.5 U ADH supposedly bound at tubular pore sites was too small to be detected with our bioassay.3. Under the assumption that 1 U Pitressin was used up for the reabsorption of approximately 4 cm³ water a vasopressin-water-equivalent in the order of 1 mole vasopressin for 10⁸ mole water reabsorbed, could be calculated.4. The amount of vasopressin excreted by the kidney follows an exponential function with a half life of 5 min.5. The vasopressin clearance is approximately 1.0 cm³/min · rat and lies within the range of inulin clearance (1.2 cm³/min · rat). It is suggested that elimination of excess vasopressin proceeds by a simple filtration process.6. Calculating on a weight basis the ADH-requirement of the 200 times heavier human kidneys leads to the value 200–300 U. Using a vasopressin-water-equivalent of 4–5 cm³ water per 1–1.5 U (action time 50 min) it can be predicted that the human kidney must lose approximately 261 water per day under the condition of a complete lack of vasopressin. This agreement with the actual observations in diabetes insipidus patients supports the belief that some of the concepts worked out in the alcohol anesthetized rat are valid under circumstances other than the strict conditions of this preparation.

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Zusammenfassung In früheren Experimenten wurden Einzelinjektionen von 0,2–10 E ADH in Alkohol narkotisierte Ratten gemacht und die Wassermenge bestimmt, die während der antidiuretischen Phase rückresorbiert wurde. Die Dosis-Antwort-Kurve hatte den Charakter einer Sättigungskurve mit einem praktisch linearen Anstieg bis 1–1,5 E. Der antidiuretische Effekt war bei einer Injektion von 4–6 E nicht deutlich größer als bei der kleineren Dosis, gleichzeitig fanden sich 75% der injizierten ADH-Menge im Urin der antidiuretischen Phase. Es wurde geschlossen, daß während einer Einzelinjektion von ADH 1–1,5 E fast augenblicklich von Receptoren der Tubuluswand gebunden und dann während des langssamen Prozesses der Wasserresorption inaktiviert werden, während das überschüssige ADH im Urin ausgeschieden wird. Schätzungsweise war 1 E ADH erforderlich, um 5 cm³ Wasser rückzuresorbieren. Die für den Resorptionsvorgang erforderliche Zeit war relativ kurz bei hoher Resorptionsrate und umgekehrt.In den vorliegenden Untersuchungen wurden die Einzelinjektionen mit 20 E wiederholt. Die höhere Dosis erlaubte, die antidiuretische Aktivität (ADA) in drei aufeinanderfolgenden Urinportionen der antidiuretischen Phase getrennt zu bestimmen. Das Resultat bestätigte die Arbeitshypothese.1. Die mit 20 E Pitressin erzielte ADA von 4 cm³ war nicht größer, sondern (nicht signifikant) kleiner als diejenige, die in den früheren Versuchen mit 5 E Pitressin oder 1 E Tonephin gefunden wurden.2. 95±15% der injizierten ADA erschienen im Urin. Das heißt, die Differenz zwischen den 20 injizierten E und den 18,5–19 E, die nach Abzug der vermutlich an der Tubuluswand gebundenen 1–1,5 E ausgeschieden wurden, war zu klein, um mit unserem Bioassay entdeckt zu werden.3. Unter der Annahme, daß 1 E Pitressin verbraucht wurde für die Resorption von ungefähr 4 cm³ Wasser, kann ein Vasopressin-Wasser-Äquivalent von ungefähr 1 Mol Vasopressin für 10⁸ Mol Wasser errechnet werden.4. Die durch die Niere ausgeschiedene Vasopressin-Menge folgt einer e-Funktion mit einer Halbwertszeit von 5 min.5. Die Vasopressin-Clearance beträgt ungefähr 1,0 cm³/min · Ratte und liegt somit wenig unter der Inulin-Clearance (1,2 cm³/min · Ratte). Diese Tatsache legt die Vermutung nahe, daß das überschüssige Vasopressin durch einfache Filtration ausgeschieden wird.6. Wenn man unter Berücksichtigung der unterschiedlichen Nierengewichte den ADH-Bedarf der 200mal schwereren Menschenniere schätzt, so kommt man auf 200–300 E. Setzt man ein Vasopressin-Wasser-Äquivalent von 4–5 cm³ pro 1–1,5 E ADH bei einer Wirkzeit von 50 min in Rechnung, so sollte die menschliche Niere ca. 261 Wasser pro Tag bei völligem Fehlen von ADH verlieren. Die Übereinstimmung mit dem wirklichen Wert könnte dafür sprechen, daß ein Teil der Resultate, die an der Alkohol-narkotisierten Ratte gewonnen wurden, nicht nur unter den strikten Bedingungen dieses Präparates gelten.

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This work was supported by Contract AF 61 (052)-947 of the USAF School of Aerospace Medicine, European Office of Aerospace Research (OAR), U.S. Air Force and the Deutsche Forschungsgemeinschaft.