Adrenaline-stimulated,aspirin-sensitive synthesis of histamine in the rat

The intravenous injection of 40 g/kg of adrenaline raised total rat lung histamine from 5.3±0.7 g, to 8.4±0.7 g and rat skin histamine from 624±51 g to 835±85 within 5 min. These changes were no longer apparent after 10 min. Stomach histamine was unaffected. Blood drawn 2 min after the injection of adrenaline failed to show an increased content of histamine. Rats given 1 mg/kg of compound 48/80, had greatly elevated levels of histamine in blood, but exhibited no increase in lung histamine. This result, as well as the extent of the increase of histamine observed in skin, which cannot be accounted for in any other way, point towards stepped-up local synthesis as the origin of the effect of adrenaline. Aspirin (20 mg/kg, intravenously 10 min prior to adrenaline), prevented increases of skin histamine. Evidence suggesting mast cells as the site of action of adrenaline, is discussed.     

Neuromuscular transmission in experimental autoimmune myasthenia gravis (EAMG)

Chronic experimental autoimmune myasthenia gravis (EAMG) was induced in rats by immunization with acetylcholine receptor (AChR) purified from the electroplax of Torpedo californica. 35–40 days after immunization, serum anti-AChR antibody titers were about 40 nM. At this stage, electrophysiology was performed on isolated M. omohyoideus muscle-preparations from myasthenic and from normal (control) rats.For the study of the equilibrium interaction between acetylcholine (ACh) and AChR, dose-response curves were obtained by quantitative ionophoretic application of ACh to voltage-clamped end-plates. Analysis of dose-response curves yielded the following parameters: maximum end-plate conductance per unit surfaceg _max (EAMG)=10.3±1.1 nS/m²,g _max (normal)=20.2±1.8 nS/m²; apparent dissociation constant K (EAMG)=96±5 M, K (normal)=58±6 M; Hill-coefficient n_H (EAMG)=2.3±0.1, n_H (normal)=2.3±0.1. Single channel properties were derived from an analysis of ACh-induced end-plate current noise: the mean single channel conductance was (EAMG)=29.1±2.2 pS, (normal)=27.6±1.8 pS and the mean channel life-time (EAMG)=1.39±0.09 ms, (normal)=1.32±0.08 ms (T=22°C).The electrophysiological data are interpreted as follows: (1) At myasthenic end-plates there is a 50–60% reduction of functioning AChR (decrease ofg _max). A total number of about 2×10⁶ (1×10⁶) channels per end-plate was calculated for control (myasthenic) rats. (2) The affinity of AChR for ACh is reduced and/or there is an impediment of the conformational change from the closed- to the open-channel configuration (increase of K). (3) Single channel properties are essentially unaffected.This work was supported by the Deutsche Forschungsgemeinschaft SFB 38, project N and We 667/6     

Plasma histamine and clinical tolerance to infused histamine in normal,atopic and urticarial subjects

Five subjects with a recent history of urticaria (U), five atopic (A) subjects and a non-atopic (NA) control group were given intravenous infusions of histamine starting at 0.05 g/kg/min, increasing by 0.05 g/kg/min every 30 minutes to a maximum of 0.35 g/kg/min. Plasma histamine levels were monitored every 15 minutes. The infusion was stopped when an objective clinical endpoint was reached, involving either evidence of peripheral vasodilatation (rise of skin temperature by at least 1 °C) or a 20% fall of peak expiratory flow rate.There were no significant differences in resting plasma histamine in the three groups. Those with urticaria reached the clinical endpoint at a lower infusion rate than non-atopic subjects (U 0.22±0.02 g/kg/min; A 0.26±0.02 g/kg/min; NA 0.32±0.2 g/kg/min.p<0.008) they also received a lower total histamine dose (U 1.12±0.33 mg; A 1.42±0.38 mg, NA 2.2±0.51 mg,p<0.008). Atopic subjects with a history of asthma, eczema or rhinitis also tolerated histamine poorly, some subjects reaching a clinical endpoint while the plasma histamine level was still relatively low (U 1.52±0.4 ng/ml, A 0.85±0.19 ng/ml, NA 1.4±0.44 ng/ml,p=0.05). After the histamine infusion was stopped, the fall in the blood level of histamine was slower in urticarial subjects than in the other two groups, with a half-life of 6.2±1.3 min (A 3.0±1.2 min, NA 4.0±0.7 min,p<0.02). There were thus differences in the metabolism of histamine in our non-atopic urticarial subjects and increased histamine sensitivity in atopic subjects which require further study.     

Tubular transport processes in proximal tubules of hypothyroid rats. Lack of relationship between thyroidal dependent rise of isotonic fluid reabsorption and Na+−K+-ATPase activity

Hypothyroid rats reconstituted with 10 g/kg b.w. per day of tri-iodothironine (T₃) for 4 days resulted in normal free T₃ and TSH levels. FT₃ levels were: 0.53±0.3 pg/ml in hypothyroid rats; 2.78±1.21 pg/ml in hormone reconstituted rats and 2.90±0.90 pg/ml in euthyroid rats. TSH levels were 3,508±513 g/ml in hypothyroid rats; 1,008±204 g/ml in reconstituted rats and 270±184 ng/ml in euthyroid rats.When hypothyroid rats were reconstituted with 50 g T₃/kg b.w. per day, TSH levels were nearly normal after 4 days (1,157±621 ng/ml). However FT₃ levels after 1–4 days were always higher than in euthyroid rats.Hypothyroid rats show a decrease in isotonic fluid reabsorption (J _v) in the proximal tubule (1.50±0.08 versus 4.96±0.23 10^–2 nl·mm^–1·s^–1 in euthyroid animals). 1 day after T₃ (10 g/kg b.w./day) injectionJ _v was increased significantly to 2.05±0.20 10^–2 nl·mm^–1·s^–1 and continued to increase during 4 days of T₃ reconstitution.When 50 g T₃/kg b.w./day was used,J _v increased to 2.75±0.07 after 1 day and to 3.10±0.42 10^–2 nl·mm^–1·s^–1 after 4 days.J _v was never reaching a value close to that of euthyroid rats because the tubular radius in hypothyroid rats (14.7±1.8 m) is less than that of euthyroid rats (19.2±0.5 m). The radius in hypothyroid rats treated with T₃ was unchanged over a 4 day course with either high or low doses of T₃.Na⁺–K⁺-ATPase activity was found to be 2.91±0.16 M P_i/h×mg protein in homogenates of kidney cortex from hypothyroid rats. Treatment of hypothyroid rats with 10 g or 50 g of T₃ resulted in an initial decrease in ATPase activity, followed by an increase to base level in hypothyroid rats with 10 g and a significantly higher level with 50 g. This decrease in ATPase activity was contrasted to the increase inJ _v.These data indicate that there is a dissociation between the effects of physiological doses of thyroid hormones on proximal tubular reabsorption and the effects of T₃ on Na⁺–K⁺-ATPase activity of kidney cortex. This leads to question the relationship between sodium transport and ATPase activity under physiological doses of thyroid hormones. An early effect of physiological doses of thyroid hormones on brush border Na⁺ permeability is suggested.     

Caryospora najadae sp.n. (Apicomplexa: Eimeriidae) from Dahl's whip snake,Coluber najadum (Serpentes: Colubridae)

Oocysts of a newCaryospora species,Caryospora najadae, are described from the feces of a Dahl's whip snake,Coluber najadum, from Israel. The spherical oocysts ofC. najadae measure 31.9(27.9–36.3) m in diameter and lack a micropyle and a oocyst residuum. The oocyst wall is between 1.5–2 m thick. The ovoid sporocysts are 15.2(14.0–16.4) m wide and 21.1(19.9–22.2) m long. A sporocyst residuum, a Stieda body and substieda body are present. The sporulation is completed in about 72 h at 21.1±2° C. Sporozoites are elongate measuring circa 19–21×2–2.5 m.     

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.     

Control of recombination within and between DNA plasmids of Saccharomyces cerevisiae

Summary [2 m⁺ and [2m°] yeast were transformed to stable leucine prototrophy with the hybrid yeast — E. coli plasmid, pJDB219. This plasmid contains the entire sequence of the endogenous 2 m yeast DNA plasmid in addition to the yeast nuclear LEU2 ⁺ gene and the Co1E1 derivative, pMB9. In the [2 m⁺] transformants, a new wholly yeast LEU2 ⁺ plasmid, pYX, was generated, probably by a recombination event between pJDB219 and 2 m DNA. The plamid, pYX, in the absence of 2 m DNA, was found to exist in equimolar amounts of two forms, A and B, which probably arise by intramolecular recombination across the inverted repeat sequences of the 2 m DNA portion of the plasmid. pJDB219 was found to require the presence of 2 m DNA to undergo this intramolecular recombination. The results suggest that 2, m DNA and pYX code for a gene product required in this recombination event which pJDB219 cannot produce.     

2,6-Diamino-N-([1-(1-oxotridecyl)-2-piperidinyl]methyl)hexanamide (NPC 15437): A selective inhibitor of protein kinase C

NPC 15437 inhibited protein kinase C (PKC) activity and [³H]phorbol 12,13-dibutyrate (PDBu) binding to the enzyme in a concentration-dependent manner (IC₅₀ values, 19±2 M and 23±4 M, respectively). No inhibition of cAMP-dependent protein kinase A (PKA) or calcium/calmodulin-dependent myosin light chain kinase (MLCK) was observed. A detailed kinetic analysis of the interaction of NPC 15437 and a homogeneous preparation of PKC-alpha revealed a competitive type of inhibition with respect to activation of the enzyme by both phorbol 12-myristate 13-acetate (PMA) (K _i =5±3 M) and phosphatidylserine (PS) (K _i =12±4 M). Mixed inhibition (predominantly of the non-competitive type), with respect to activation of the enzyme by calcium, was also observed. These studies indicate that NPC 15437 is a selective inhibitor of PKC, interacting at the regulatory region of the molecule. NPC 15437 inhibited phorbol ester-induced ear edema in mouse (IC₅₀=175 g/ear) demonstrating the ability of NPC 15437 to inhibit PKC-mediated activity in intact cells.     

The activation of protein kinase C prevents PGE2-induced inhibition of AVP-dependent cAMP accumulation in the rat outer medullary collecting tubule

Previous studies have demonstrated that prostaglandin E₂ (PGE₂) inhibits arginine vasopressin-(AVP)dependent adenosine 3,5-cyclic monophosphate (cAMP) accumulation in microdissected rat outer medullary collecting tubules (OMCD), by a mechanism unrelated to the inhibition of cAMP synthesis. The potential role of the activation of protein kinase C (PKC) to explain the negative regulation elicited by PGE₂ was investigated in this study. Single OMCD samples were pre-incubated (10 min, 30°C) in the presence or absence of either activators of PKC, phorbol 12-myristate 13-acetate (PMA), 1-oleoyl-2-acetyl-glycerol (OAG), dioctanoylglycerol (DOG) or an inhibitor of PKC, staurosporine (SSP). These compounds were present also with the agonists tested during the incubation period (4 min, 35°C). In contrast to PGE₂, activators of PKC did not decrease AVP-dependent cAMP accumulation (mean ±SEM): 1nM AVP=47.1±6.8 fmol · mm^–1· 4 min^–1; AVP + 0.3 M PGE₂=20.1±2.7, P<0.01 versus AVP; AVP + 10 nM PMA=42.0±4.7, NS versus AVP; AVP + 50 g/ml OAG=44.1±4.8. NS versus AVP, N= 5 experiments. However, 10 nM PMA prevented PGE₂-induced inhibition: AVP + PGE₂= 44.2±3.5% of the response to AVP and 90.3±3.2% without and with PMA respectively, N= 16. Similar results were obtained with either 50 g/ml OAG or 25 g/ ml DOG (AVP + PGE₂ + OAG=92.9±6.6% of the response to AVP, N= 8; AVP + PGE₂ + DOG=94.1 ±5.3%, N= 7). OAG, DOG, PMA or PMA + PGE₂ had no intrinsic agonist activity in the rat OMCD and the addition of an inactive phorbol ester did not prevent PGE₂-induced inhibition. SSP, 50 nM or 0.1 M, did not affect the inhibition due to PGE₂ but abolished the reversion by PMA of PGE₂-induced inhibition. A similar regulation was observed on forskolin-(FK)dependent cAMP accumulation: 5 M FK + 0.3 M PGE₂= 37.7±6.2% of the response to FK; FK + PGE₂ + 10 nM PMA=89.5±6.7%; FK + PGE₂ + PMA + 0.1 M SSP=43.1±7.9%, N= 4. The inhibition induced by an ₂-adrenergic agonist, clonidine 1 M, was not blocked by the activation of PKC. In fura-2-loaded OMCD samples, 10nM PMA decreased by 63.3±5.0% and by 57.2±7.1% the peak and plateau phases, respectively, of the increase in intracellular calcium concentration ([Ca²⁺]_i) obtained with PGE₂ when compared to control responses in the same tubules (n=12) and did not affect the increase in [Ca²⁺]_i induced by 0.1 mM carbachol. It is concluded that: (1) in the rat OMCD the activation of PKC by PMA or analogues of diacylglycerol did not reproduce PGE₂-induced inhibition of AVP- or FK-dependent cAMP accumulation, but prevented specifically this inhibitory action; and (2) this reversion might be the consequence of the effect of PKC activation which impaired the rises in [Ca²⁺]_i induced by PGE₂.     

Life cycle ofHepatozoon mehlhorni. sp. nov. in the viperEchis carinatus and the mosquitoCulex pipiens

Hepatozoon mehlhori sp. nov. and its developmental stages from the tissues of the Egyptian viperEchis carinatus and the mosquitoCulex pipines are described. The erythrocytic parasites were differentiated into the small form (trophozoite) measuring 14.5±0.6×4±0.12 m and the mature form (gametocyte) measuring 17.2±1.6×5.4±0.5m. Merogony took place in the pulmonary endothelial cells and in the parenchyma cells of the liver and spleen of the infected vipers. Two types of meront were found. The large meronts (macromeronts) were 30.2±1.73×22.6±1.2 m in size and yielded 16–40 (average 28) micromerozoites measuring 17.2±0.7×5±0.15 m. The small, meronts (micromeronts) measured 18.2±0.6×13.5±0.5 m and yielded 2–14 (average, 8) macromerozoites that were 15.1±0.12×6.2±0.8 m in size. After syzygy in the haemocoel of the mosquito, the microgamont produced four uniflagel-late microgametes (6.4±0.3×4.5±0.5 m in size, with a short flagellum measuring 3.2±0.1 m); on the 3rd day post-infection (p.i.)., one of these fertilized the macrogamete, giving rise to the zygote. The oocyst developed from the zygote on the 5th day p.i. and measured 135±2.6×120±1.8 m. About 11–60 (average, 35) sporoblasts were formed by centripetal invaginations from each oocyst on the 8th day p.i. and developed into sporocysts on the 14th day p.i. Inside each sporocyst, 5–12 (average, 8) sporozoites, each measuring 12.6±1.2×4.1±0.3 m, developed on the 16th day p.i. According to the above-mentioned characteristics the parasite was recorded as being a new species and was namedHepatozoon mehlhorni. Experimental transmission was accomplished by i.p. inoculation of the infectious stages (sporozoites) into uninfected vipers and led to the appearance of blood stages at 4–6 weeks p.i.Abbreviations BLC Blood capillary - DMS developing merozoites - DSP developing sporoblasts - E erythrocyte - F flagellum - HC host cell - HN host nucleus - M Meront - MA macrogamont/macrogamete - MG Microgamete - MIG microgamont - MS merozoites - N nucleus - NG micleus of the microgamete - OC oocyst - P erythrocytic parasite - PV Parasitophorous vacuole - SP sporoblast (s) - SPC sporocyst (s) - SPR sporozoite (s) - ZY zygote (young oocyst)     

Inhibition of tumor necrosis factor in the brain suppresses rabbit sleep

Tumor necrosis factor (TNF) is a cytokine that possesses many biological activities, including enhancement of non-rapid-eye-movement sleep (NREMS). The role of endogenous TNF in the regulation of spontaneous sleep is unknown. If TNF is involved in sleep regulation, then reduction of endogenous TNF should suppress spontaneous sleep. A soluble TNF-binding protein I (TNF-BP I) and a synthetic fragment of TNF-BP I, TNF-R-(159–178), that contains the biologically active region of TNF-BP I, were used. These substances bind TNF and possess TNF-inhibitory activity; their effects on rabbit sleep after intracerebroventricular injection were determined across a 6-h recording period. Two doses of TNF-BP I (0.05 g and 0.5 g) were administered; the higher dose of TNF-BP I significantly decreased NREMS. Four doses of TNF-R-(159–178) (0.25 g, 2.5 g, 25 g and 50 g) were used. The 25 g and 50 g doses significantly suppressed NREMS. The highest dose (50 g) also decreased REM sleep. These results are consistent with the hypothesis that endogenous brain TNF is involved in the regulation of normal sleep.     

A pharmacological study of the control of nasal cooling in the dog

Summary Clearance studies were performed in order to examine the effect of expansion of extracellular fluid volume (ECFV) on the maximal reabsorptive capacity for inorganic phosphate (Tm_Pi) in acutely parathyroidectomized (PTX) and intact rats. Tm_Pi values were obtained in both control and volume expansion. In PTX rats, the Tm_Pi values in control and expansion were 8.25±1.52 and 6.14±1.02 mol/min (mean values ±S.D.), respectively; the Tm_Pi/ GFR values were 2.96±0.31 and 2.09±0.30 mol/ml, respectively. Inintact rats, the Tm_Pi values in control and expansion were 3.56±0.94 and 2.98 ±0.94 mol/min, respectively, and the Tm_Pi/GFR values were 1.34±0.23 and 1.05±0.23 mol/ml, respectively. From these results it is concluded that expansion of ECFV decreases the Tm_Pi values both in the absence and presence of parathyroid hormone.This work was supported in part by a grant from the Deutsche Forschungsgemeinschaft (Fr 239/5)     

Sodium salicylate: alternate mechanism of central antipyretic action in the rat

Infusion of sodium salicylate (50.0 or 100.0 g/l) into the ventral septal area (VSA) of the rat brain suppressed Prostaglandin-E₁-induced hyperthermia. Infusion of artificial cerebrospinal fluid (aCSF) or 10.0 g doses of salicylate did not. The suppression of intracerebroventricularly-induced (icv) Prostaglandin E₁ (PGE₁) hyperthermia was not due to a hypothermic action of salicylate since salicylate infusions given during cold exposure (10.0°C) did not lower core body temperatures. A possible interaction between salicylate and endogenous arginine vasopressin (AVP) was investigated. Infusion of both salicylate (50.0 g/l) and either AVP antiserum or AVP antagonist into the VSA resulted in PGE hyperthermias occurring at levels which were not different from control levels as opposed to enhanced hyperthermia (antiserum or antagonist alone) or suppressed hyperthermia (salicylate alone). These results are consistent with the notion that sodium salicylate infusions within the VSA enhance AVP action and thus bring about the attenuation of PGE-induced hyperthermia.     

Clinical,hematologic, and immunologic effects of interleukin-10 in humans

We conducted a double-blind, placebo-controlled study to investigate the safety, pharmacokinetics, and immunological properties of interleukin-10 (IL-10) administration in healthy humans. Volunteers received a single intravenous bolus injection of recombinant human IL-10 (1, 10, or 25g/kg) or placebo. Cytokine production in whole blood and peripheral blood mononuclear cells (PBMC) was assessed before and 3, 6, 24, and 48 hr after the injection. Peak serum concentrations of IL-10 (15±1.1, 208±20.1, and 505±22.3 ng/ml) occurred after 2–5 min for 1, 10, and 25g/kg IL-10, respectively. The terminal-phase half-life was 3.18 hr. A transient leukocytosis (24–63% above baseline) was observed 6 hr after injection, which coincided with a dose-dependent decrease (12–24%) in neutrophil superoxide generation. There was a marked inhibition (60–95%) of endotoxin-induced IL-6 production from whole blood in each group receiving IL-10. Production of IL-8 in endotoxin-stimulated blood was reduced in the 10g/kg group. In PBMC stimulated with phytohemagglutinin and phorbol ester, there was a decrease (72–87%) in interferon- (IFN) production 6 hr after IL-10 with a return to pre-IL-10 levels after 24 hr. This reduction was only partially associated with a decrease in the number of CD2-bearing cells. We conclude that IL-10 administration into humans is without significant side effects, and a single injection reducesex vivo production of IL-6, IL-8, and IFN.     

The dual effects of ouabain on45Ca2+ transport and contractility in adult rat ventricular myocytes

We used isolated ventricular myocytes to study⁴⁵Ca²⁺ transport in the presence of three concentrations of ouabain (10 nM, 1 M, and 100 M) in Tyrode solution containing 1 mM CaCl₂. The cells were quiescent and during⁴⁵Ca²⁺ uptake and⁴⁵Ca²⁺ efflux experiments 10 nM ouabain decreased Ca²⁺ content, 1 M, didn't change it appreciably, and 100 M increased it significantly. Qualitatively, the same results were obtained at 22°C and 35°C. Ouabain did not significantly affect the electrical activity of isolated, electrically stimulated myocytes, but it increased the amplitude of shortenings of these myocytes in a dose-dependent manner. Thus, the positive inotropic effect of ouabain at therapeutic doses (10 nM) occurs in spite of decreased Ca²⁺ content, while at high toxic doses the positive inotropic effect is accompanied by an increment in Ca²⁺ content. These data support the hypothesis that the mechanisms of positive inotropy of ouabain are different at therapeutic and toxic concentrations of this drug. Finally, our study demonstrates that the effects of low doses of ouabain are independent of the release of endogenous catecholamines.     

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.     

Effect of depolarizing and hyperpolarizing agents on the membrane potential difference of primary cultures of rabbit aorta vascular smooth muscle cells

Vascular smooth muscle cells of rabbit aorta were enzymatically dispersed, kept in primary culture, and studied between days 1 and 7 in a bath rinsed with Ringer-like solution at 37°C. The electrical membrane potential difference (PD) was measured with microelectrodes. The mean value of PD was –50±0.4 mV (n=53). Cromakalim (BRL 34915), 1 mol/l and 10 mol/l, hyperpolarized the membrane potential by 9±1 mV (n=11) and 15±1 mV (n=53) respectively. Glibenclamide (10 mol/l) abolished the hyperpolarizing effect of cromakalim (n=6). Simultaneous addition of cromakalim and glibenclamide (both 10 mol/l, n=11) and glibenclamide itself (10 mol/l, n=7) had no effect on PD. In patch-clamp experiments in outside-out-oriented Ca²⁺-sensitive K⁺ channels, cromakalim increased the open probability (P _o) only slightly and only with a cytosolic Ca²⁺ activity of 1 mol/l. In all other series cromakalim had no effect on the P _o of these channels. Forskolin (10 mol/l) hyperpolarized PD by 6±1 mV (n=13). The nucleotides UTP, ATP and ITP (10 mol/l) depolarized PD by 12±1 mV (n=7), 8±1 mV (n=65) and 5±1 mV (n=6) respectively. GTP, [,-methylene]ATP and adenosine had no significant effect. Mn²⁺ (1 mmol/l, n=18), Ni²⁺ (1 mmol/l, n=13), Co²⁺ (1 mmol/l, n=11), Zn²⁺ (1 mmol/l, n=6) and the Ca²⁺-channel blockers verapamil and nifedipine (both 0.1 mmol/l, n=6) did not attenuate the depolarization induced by 10 mol/l ATP. Fetal calf serum (100 ml/l, n=7) depolarized PD by 11±2 mV. This effect was not abolished by nifedipine or by replacing NaCl by choline chloride. The data indicate that PD of vascular smooth muscle cells is depolarized by P₂ agonists and hyperpolarized by the K⁺-channel opener cromakalim. The effect of cromakalim is antagonized by glibenclamide. The effect of cromakalim is probably not mediated by the K⁺ channel identified in excised patches.Supported by DFG Gr 480/10     

Transient improvement of polymorphonuclear leukocyte function by splenectomy in β-thalassemia

Host defense mechanisms in transfusion-dependent non-splenectomized patients with -thalassemia were studied. Polymorphonuclear leukocytes (PMNLs) of non-splenectomized patients responded poorly to zymosan generated chemotactic factors. Chemotactic indices were 22.1 m ± 2.8 (mean ± S.D.) using zymosan activated serum (ZAS) as the attractant in comparison to 20.4 m ± 2.6 when fresh untreated serum was used. In contrast, chemotactic indices of normal PMNLs increased from 21.1 m to 33.6 m ± 3.1 in response to ZAS. Normal PMNL responses to a mixture of normal ZAS and thalassemic serum were inhibited; the mean chemotactic index was 18.1 m ± 5.1 with use of ZAS alone. Splenectomy temporarily reverses these alterations. Adherence to nylon wool of PMNLs suspended in fresh thalassemic serum prior to splenectomy was 3.1% ± 1.1 (mean ± S.D.); 20 days after splenectomy adherence increased to 14.0% ± 2.8 (P = 0.0001) and remained at this level for 90 days. At 120 and 150 days after splenectomy adherence decreased to 1.5% ± 0.8 and 1.0% ± 0.85 respectively. Splenectomy also transiently abrogated the failure of zymosan to generate chemotactic factors in thalassemic serum.This study was presented in part at the American Federation for Clinical Research, Central Society, Infectious Diseases, Chicago, Illinois, November 3, 1983     

Modulation of bradykinin responses in airway smooth muscle by epithelial enzymes

We have studied the effect of epithelium removal on responses of guinea pig trachea to bradykinin (BK). BK (1 nM–10 M) gave a concentration-dependent relaxation when epithelium was present (E+: EC₅₀=10±3 nM). Epithelium removal resulted in a biphasic response to BK with relaxation at low concentrations (E–: EC₅₀=3.0±1.0 nM) and a recontraction to baseline at higher concentrations (EC₅₀=2.0±1 M). Phosphoramidon (10 M), an inhibitor of neutral endopeptidase (NEP), which cleaves BK into inactive peptides, potentiated relaxation (EC₅₀=1.0±0.9 nM and 0.1±0.1 nM in E+ and E respectively) and contraction in trachea with intact epithelium (EC₅₀=0.08±0.03 M). Inhibition of cyclooxygenase by indomethacin (5 M), inhibited relaxation to BK in E+ tracheal segments, resulting in a slight contraction (EC₅₀=1.0 M), whereas a potent contractile response was observed in E–segments (EC₅₀ 1.6 M, maximal contraction >1 g). In the presence of both indomethacin and phosphoramidon BK caused contraction, even in the presence of epithelium (EC₅₀=0.2±0.11 M), and the response in the absence of epithelium was similar to the response observed in trachea with intact epithelium (EC₅₀=0.25±0.1 M). The contractile effect of BK on airway smooth muscle may be inhibited by a protective role of epithelium, due to release of relaxant prostanoids and by degradation by epithelial NEP. In asthma, bronchoconstrictor responses to BK may be partly explained by loss of airway epithelium.     

Macromolecular structure of axon membrane and action potential conduction in myelin deficient and myelin deficient heterozygote rat optic nerves

Summary The macromolecular structure of the axon membrane in optic nerves from 25-day-old male littermate control and myelin deficient (md) rats and 16-month-old md heterozygotic rats was examined with quantitative freeze-fracture electron microscopy.The axon membrane of control optic nerves displayed an asymmetrical partitioning of intramembranous particles (IMPs); P-fracture faces of myelinated internodal axon membrane were more particulate than those of pre-myelinated axons (1600 1100 m^–2, respectively), while relatively few IMPs (150 m^–2) were present on external faces (E-faces) of internodal or pre-myelinated axon membrane. Amyelinated axons of md optic nerves also exhibited an asymmetrical partitioning of IMPs; protoplasmic membrane face (P-face) IMP densities, taken as a group, exhibited a wide range (600–2300 m^–2) and, in most regions, E-faces displayed a relatively low IMP density (175 m^–2). Axons of > 0.4 m diameter exhibited significantly greater mean P-face IMP density than axons < 0.4 m diameter. Aggregations of E-face IMPs (350 m^–2) were occasionally observed along amyelinated axon membrane from md optic nerves.Optic nerves from md heterozygote rats exhibit myelin mosaicism, permitting examination of myelinated and amyelinated axon membrane along the same tract. The axon membrane exhibits different ultrastructure in these two domains. Myelinated internodal axon membrane from md heterozygote optic nerves exhibits similar P- and E-face IMP densities to those of control internodal axolemma (1800 and 140 m^–2, respectively). Amyelinated axons in the heterozygote exhibit a membrane structure similar to amyelinated axons in md optic nerve. P-face IMP density of large diameter (> 0.4 m) amyelinated axons from md heterozygote optic nerves is significantly greater than that of small calibre (< 0.4 m) axons. In most regions, amyelinated axon membrane exhibits a relatively low E-face IMP density (200 m^–2); however, focal aggregations (400 m^–2) of E-face particles are present.Electrophysiological recordings demonstrate that amyelinated axons in md optic nerves support the conduction of action potentials. Compound action potentials in md optic nerves exhibit a monophasic configuration, even at 20-days postnatal, similar to that of pre-myelinated optic nerve of 7-day-old normal rats. Moreover, conduction velocities in the amyelinated 20-day-old md optic nerve are similar to those displayed by pre-myelinated axons from 7-day-old optic nerves. These results are consistent with persistence of action potential conduction in md axons, despite the absence of myelination in the optic nerves of the md mutant.