Guanylin activates Cl− secretion into the lumen of seawater eel intestine via apical Cl− channel under simulated in vivo conditions

Guanylin (GN) action on seawater eel intestine was examined under simulated in vivo conditions, where isotonic luminal fluid has low NaCl and high MgSO4 (MgSO4 Ringer). In Ussing chamber, MgSO4 Ringer induced serosa-negative potential difference (PD) even after bumetanide treatment, which is due to the higher paracellular Na+ permeability over Cl−, as confirmed by the replacement by MgCl2 (no Cl− gradient) or Na2SO4 Ringer (no Na+ gradient). Luminal GN reversed serosa-negative PD, probably by enhancing Cl− secretion into the lumen, as the GN effect was blocked by apical Cl− channel blockers [diphenylamine-2-carboxylic acid (DPC), 5-nitro-2-(3-phenylpropylamino) benzoic acid, glibenclamide but not cystic fibrosis transmembrane regulator (CFTR)inh-172] or replacement of luminal fluid by MgCl2 Ringer. The blockers' effect was undetectable when normal Ringer was on both sides. In the sac preparation, NaCl secretion occurred into the lumen (Na+ > Cl−), and GN further enhanced Cl− secretion (Cl− > Na+), resulting in water secretion. These GN effects were also blocked by DPC. Quantitative analyses showed that isotonic NaCl is absorbed when luminal fluid is normal Ringer, but, when luminal fluid is MgSO4 Ringer, hypertonic NaCl, almost equivalent to seawater, is secreted into the lumen after GN. These results indicate that GN stimulates the secretion of hypertonic NaCl into the lumen of seawater eel intestine, like rectal gland of marine elasmobranchs, to get rid of excess NaCl although marine teleost intestine is thought to have only absorptive-type cells with a unique Na-K-Cl cotransport system. The secreted NaCl may activate the cotransport system and further help absorb water in the final segment of seawater eel intestine.

Nitro-L-arginine attenuates hypercapnic cerebrovasodilation without affecting cerebral metabolism

We have previously demonstrated that topical cortical application of nitro-L-arginine (L-NA), a potent inhibitor of nitric oxide (NO) synthesis, attenuates resting cerebral blood flow (CBF) and the cerebrovasodilation elicited by hypercapnia. In this study, we sought to determine whether these cerebrovascular effects of L-NA are secondary to a depression in cerebral metabolism. Rats were anesthetized (chloralose, 80 mg/kg) and artificially ventilated. Arterial pressure and blood gases were monitored. The frontal cortex was exposed and superfused with normal Ringer (pH 7.3-7.4; 37 degrees C) or with Ringer containing L- or D-NA. CBF or cerebral glucose utilization (CGU) was measured autoradiographically using the [14C]iodoantipyrine or 2-[14C]deoxy-D-glucose method, respectively. Application of normal Ringer did not affect CBF at the site of superfusion (n = 5; P > 0.05, paired t test). Application of L-NA (1 mM; n = 5), but not D-NA (1 mM; n = 6), attenuated resting CBF by 33 +/- 5% (P < 0.05; analysis of variance). During hypercapnia (partial pressure of CO2 = 55-60 mmHg), L-NA attenuated the CBF increase by 78 +/- 6% (n = 5/group; P < 0.05 from Ringer), whereas D-NA had no effect (P > 0.05). Resting CBF and the CBF response to hypercapnia were largely unaffected in brain regions outside the field of superfusion. In contrast to hypercapnia, L-NA (1 mM) did not attenuate the increases in CBF elicited by topical application of papaverine (10-1,000 microM; n = 8).(ABSTRACT TRUNCATED AT 250 WORDS)

Segmentally distinct effects of depolarization on intracellular [Ca2+] in renal arterioles

Experiments were performed to determine the influence of depolarization on intracellular Ca2+ concentration ([Ca2+]i) in renal arterioles and the possible role of voltage-gated Ca2+ channels in these responses. Glomeruli with attached arterioles and thick ascending limb were dissected from rabbit kidney and loaded with fura 2. [Ca2+]i of nonperfused arterioles was monitored using a microscope-based dual-excitation wavelength spectrofluorometry system. Afferent arteriolar [Ca2+]i averaged 150 +/- 11 nM (n = 20) when bathed in Ringer solution containing 1.5 mM Ca2+ and 5 mM K+. Replacement of the normal Ringer solution with one containing 100 mM K+ significantly increased afferent arteriolar [Ca2+]i to 196 +/- 12 nM. This response was abolished in the absence of extracellular Ca2+. In the presence of 1 microM nifedipine, 100 mM K+ elicited a 10% decrease in afferent arteriolar [Ca2+]i (P < 0.05). Thus nifedipine reversed the afferent [Ca2+]i response to depolarization, implicating voltage-gated Ca2+ channels as the influx pathway. In contrast to the behavior of afferent arterioles, the 100 mM K+ solution reduced efferent arteriolar [Ca2+]i from 188 +/- 17 to 148 +/- 13 nM (n = 11, P < 0.01), an effect that was not influenced by nifedipine. These observations support a role for voltage-gated Ca2+ channels in eliciting depolarization-induced increases in afferent arteriolar [Ca2+]i while failing to provide evidence for operation of such a mechanism at efferent arteriolar sites.

Simultaneous measurement of Ca2+ in muscle with Ca electrodes and aequorin. Diffusible cytoplasmic constituent reduces Ca(2+)-independent luminescence of aequorin.

Estimates of cytoplasmic Ca2+ concentration ([Ca2+]i) were made essentially simultaneously in the same intact frog skeletal muscle fibers with aequorin and with Ca-selective microelectrodes. In healthy fibers under truly resting conditions [Ca2+]i was too low to be measured reliably with either technique. The calibration curves for both indicators were essentially flat in this range of [Ca2+], and the aequorin light signal was uniformly below the level to be expected in the total absence of Ca2+. When [Ca2+]i had been raised to a stable level below the threshold for contracture by increasing [K+]o to 12.5 mM, [Ca2+]i was 38 nM according to aequorin and 59 nM according to the Ca-selective microelectrodes. These values are not significantly different. Our estimates of [Ca2+]i are lower than most others obtained with microelectrodes, probably because the presence of aequorin in the cells allowed us to detect damaging microelectrode impalements that otherwise we would have had no reason to reject. The observation that the light emission from aequorin-injected fibers in normal Ringer solution was below the level expected from the Ca(2+)-independent luminescence of aequorin in vitro was investigated further, with the conclusion that the myoplasm contains a diffusible macromolecule (between 10 and 30 kD) that interacts with aequorin to reduce light emission in the absence of Ca2+.

Ultrastructural and electrophysiological changes associated with K+-evoked release of neurotransmitter at the synaptic terminals of skate photoreceptors

Bathing the skate retina in a Ringer solution containing a high concentration (100 mM) of potassium ions depolarized the visual cells, depleted the receptor terminals of synaptic vesicles, and suppressed completely the b-wave of the ERG and the intracellularly recorded response of horizontal cells (the S-potential). The depletion of synaptic vesicles was accompanied by a large increase in the extent of the plasma membrane resulting in distortion of the normal terminal profile, i.e. distension of the basal surface and elaborate infolding of protoplasmic extensions. Morphometric analysis showed that despite the changes in vesicle content and terminal structure, the combined linear extent of the vesicular and plasma membranes was unchanged from control (superfusion with normal Ringer solution); the increase in plasma membrane was equivalent to the observed loss of vesicular membrane. When returned to a normal Ringer solution, the terminals rapidly began to reform, and in about 10 min they were morphologically indistinguishable from receptor terminals seen in control preparations. After 30 min in the normal Ringer solution, the amount of membrane associated with the vesicles and the plasma membrane had reverted to control values, and once again the total membrane estimated morphometrically remained essentially the same. Thus, there is an efficient mechanism at the photoreceptor terminal for the recycling of vesicle membrane following exocytosis.The K+-induced depletion of synaptic vesicles was paralleled by a precipitous loss of responsivity in both the b-wave of the ERG and the S-potential of the horizontal cells. However, after 30-min exposure to the high K+ and a return to normal Ringer solution, the recovery of electrophysiological activity followed a much slower time course from that associated with the structural changes; 60 min or longer were required for the potentials to exhibit maximum response amplitudes. It appears that the rate-limiting step in restoring normal synaptic function following massive depletion of vesicular stores is transmitter resynthesis and vesicle loading rather than vesicle recycling.

Depression of posttetanic twitch potentiation by low calcium and calcium channel antagonists

The purpose of this investigation was to determine if antagonizing extracellular calcium influx altered posttetanic twitch potentiation (PTP). Whole muscles and muscle fiber bundles (less than or equal to 25 fibers) dissected from frog sartorius and semitendinosus muscles were mounted at optimal length in a normal Ringer solution (NR). To determine PTP, isometric twitches were evoked every 10 s (0.1 Hz) before and after a 2.5-s tetanic contraction (80 Hz). To antagonize calcium influx, low-calcium Ringer [LCR, calcium replaced by 3 mM magnesium and 1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid], NR plus diltiazem (Dilt, 30 microM), NR plus nifedipine (Nif, 10 microM), and NR plus D 600 (30 microM) were also used (n = 8 for each condition). These conditions altered pretetanic twitch tension by only -1.2 +/- 2.4, 4.2 +/- 2.3, 4.7 +/- 3.7, and 1.6 +/- 3.7% (SE), (LCR, Dilt, Nif, and D 600, P greater than 0.05) but caused a noticeable decrease in tension at the end of the tetanus. Under NR conditions, twitches evoked immediately after the tetanus were potentiated by 49.5 +/- 0.4% with the peak rate of tension development (dP/dt) increased by 44.9 +/- 0.5% (P less than 0.05). Antagonizing calcium influx depressed the PTP response by 59.8 +/- 6.2, 55.9 +/- 10.1, 73.2 +/- 6.8, and 29.8 +/- 3.6% (P less than 0.05) and increased dP/dt by 65.8 +/- 11.1, 45.7 +/- 8.6, 55.6 +/- 4.4% and 49.0 +/- 10.5% (P less than 0.05). Addition of drugs immediately after the tetanus only slightly reduced PTP but accelerated recovery of the twitch.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminophylline enhances contractility of frog skeletal muscle: an effect dependent on extracellular calcium

The effects of aminophylline (10–500 microM) on isometric twitch and tetanic forces were studied in vitro on frog semitendinosus muscle. Two hypotheses were tested: 1) that micromolar concentrations of aminophylline enhanced contractility of isolated skeletal muscle and 2) that the potentiating effect of aminophylline was dependent on the presence of extracellular calcium ions. Muscles were removed, placed in aerated Ringer solution at 20 degrees C, attached to a force transducer, and stimulated directly. Muscles in normal Ringer and aminophylline Ringer were compared throughout the frequency-force relationship from twitches to maximum tetanic force. Aminophylline increased twitch force significantly at concentrations as low as 25 microM. Over a range of stimulation frequencies, but especially at 10 and 20 Hz, aminophylline increased tetanic force. The potentiating effect of aminophylline (100 microM) was reduced or eliminated in calcium-free Ringer containing 10 mM magnesium. We conclude that aminophylline, at therapeutic concentrations, enhances muscle contractility, and the enhancement is dependent on the presence of extracellular calcium. These findings support the concept that aminophylline is effective in improving respiration in humans with airway obstruction by enhancing diaphragmatic contractility.

Chloride dependence of the HCO3 exit step in urinary acidification by the turtle bladder

To characterize the efflux of HCO-3 across the basolateral membrane of the H+-secreting cells of the turtle bladder, we examined the effect of substitution of gluconate or methyl sulfate for Cl- on the rate of acidification (JH). JH was measured as the short-circuit current in bladders in which Na+ transport was abolished with 10(-4) M ouabain. In hemibladders bathed in normal Ringer solution (Cl- = 122 mM) JH was 44.9 microA. Substitution of the Cl- resulted in a marked reduction in JH (12.5 microA with gluconate and 7.5 microA with methyl sulfate). Addition of Cl- to the mucosal surface had no effect on JH. In contrast, serosal addition of Cl- restored JH to control. The apparent Km for Cl- in gluconate Ringer was 0.13 mM. Serosal furosemide (1 mM) inhibited JH by 55% in Cl- Ringer. We conclude that HCO-3 exit across the basolateral membrane of the H+-secreting cell occurs via a Cl-HCO3 exchanger that has a high affinity for chloride.

Endplate currents in sucrose solution

Endplate currents were recorded from voltage-clamped frog muscle fibres bathed in an isotonic sucrose solution containing 2 mM K + . In this solution the major part of the current is carried by K + ions, and at negative potentials the membrane voltage–current amplitude relations of both miniature endplate currents and the single channel current estimated from noise analysis were linear, with smaller conductance than in normal Ringer solution. At positive potentials miniature endplate currents and currents induced by acetylcholine (ACh) showed a saturation, or sometimes even decline, with increasing potential. In contrast, the single channel current continued to increase linearly at these potentials. It is suggested that in sucrose solution the number of functional ACh receptors decreases as the endplate is depolarized to more positive potentials.

Intracellular calcium and desensitization of acetylcholine receptors

Acetylcholine (ACh) was applied iontophoretically to voltage-clamped endplates in frog muscle. The current induced by prolonged application of ACh decreases progressively as the membrane becomes desensitized. Desensitization was sharply localized, and at a distance of 15 μm or less the ACh sensitivity of the membrane remained normal. Desensitization still occurred in muscles exposed to Ca 2+ -free media for several hours. In these conditions the rate of desensitization was not greatly affected by altering the membrane potential. In normal Ringer (1.8 mm Ca 2+ ) desensitization was more pronounced and ACh application was frequently accompanied by localized contraction of the muscle fibre. Both the desensitization and the contraction were reduced after intracellular injection of EGTA, probably because this opposes the rise in internal Ca 2+ normally caused by ACh action.