Biophysical properties of gap junctions between freshly dispersed pairs of mouse pancreatic beta cells.

Coupling between beta cells through gap junctions has been postulated as a principal mechanism of electrical synchronization of glucose-induced activity throughout the islet of Langerhans. We characterized junctional conductance between isolated pairs of mouse pancreatic beta cells by whole-cell recording with two independent patch-clamp circuits. Most pairs were coupled (67%, n = 155), although the mean junctional conductance (gj) (215 +/- 110 pS) was lower than reported in other tissues. Coupling could be recorded for long periods, up to 40 min. Voltage imposed across the junctional or nonjunctional membranes had no effect on gj. Up to several hours of treatment to increase intracellular cAMP levels did not affect gj. Electrically coupled pairs did not show transfer of the dye Lucifer yellow. Octanol (2 mM) reversibly decreased gj. Lower concentrations of octanol (0.5 mM) and heptanol (0.5 mM) than required to uncouple beta cells decreased voltage-dependent K+ and Ca2+ currents in nonjunctional membranes. Although gj recorded in these experiments would be expected to be provided by current flowing through only a few channels of the unitary conductance previously reported for other gap junctions, no unitary junctional currents were observed even during reversible suppression of gj by octanol. This result suggests either that the single channel conductance of gap junction channels between beta cells is smaller than in other tissues (less than 20 pS) or that the small mean conductance is due to transitions between open and closed states that are too rapid or too slow to be resolved.     

Gap junction formation and functional interaction between neonatal rat cardiocytes in culture: A correlative physiological and ultrastructural study

Summary The time course of gap junction formation and growth, following contraction synchronization of cardiac myocytes in culture, has been studied in a combined (electro)physiological and ultrastructural study. In cultures of collagenase-dissociated neonatal rat cardiocytes, pairs of spontaneously beating myocytes synchronized their contractions within one beat interval within 2–20 min after they apparently had grown into contact, 45 sec after the first synchronized beat an appreciable junctional region containing several small gap junctions was already present. In the following 30 min, neither the area of individual gap junctions nor their total area increased, 75 min after synchronization both the area of individual gap junctions and their total area had increased by a factor of 10–15 with respect to what was found in the first half hour. In the period between 75 and 300 min again no further increase in gap junctional area was found. In double voltage-clamp experiments, gap junctions between well-coupled cells behaved like ohmic conductors. In poorly coupled cells, in which the number of functional gap-junctional channels was greatly reduced, the remaining channels showed voltage-dependent gating. Their single-channel conductance was 40–50 pS. The electrophysiologically measured junctional conductance agreed well with the conductance calculated from the morphometrically determined gap-junctional area. It is concluded that a rapid initial gap junction formation occurs during the 2–20 min period prior to synchronization by assembly of functional channels from existing channel precursors already present in the cell membranes. It then takes at least another 30 min before the gap-junctional area increases possibly byde novo synthesis or by recruitment from intracellular stores or from nonjunctional membranes, a process completed in the next 45 min.     

Increase in junctional conductance caused by isoproterenol in heart cell pairs is suppressed by cAMP-dependent protein-kinase inhibitor

The influence of isoproterenol (10(-6) M) on junctional conductance of cell pairs isolated from adult rat ventricle was investigated using two separated voltage-clamp circuits. The drug increased the junctional conductance by 40% (S.E. +/- 11%) (n = 10) within 20 sec. In experiments in which an inhibitor of cAMP dependent-protein Kinase was introduced into the cells the effect of isoproterenol on junctional conductance was totally suppressed. These results support the view that cAMP involved in the fast-modulation of junctional conductance in heart and that the activation of a protein Kinase is needed for the effect of the nucleotide on junctional conductance.     

Connexin32 gap junction channels in stably transfected cells: unitary conductance.

Pairs of SKHep1 cells, which are derived from a highly metastatic human hepatoma, were studied using the whole cell voltage clamp technique with patch-type electrodes containing CsCl as the major ionic species. In 12 of 81 cell pairs, current flow through junctional membranes was detectable; in the remaining 69 cell pairs, junctional conductance was less than the noise limit of our recording apparatus (worst case: 10 pS). Macroscopic junctional conductance (gj) in the small percentage of pairs where it was detectable ranged from 100 to 600 pS. Unitary junctional conductance (gamma j) determined in the lowest conductance pairs or after reducing conductance with a short exposure to the uncoupling agent halothane was 25-35 pS. To study properties of gap junction channels formed of connexin32, the parental SKHep1 cell line was stably transfected with a plasmid containing cDNA that encodes connexin32, the major gap junction protein of rat liver cells. In 85 of 98 pairs of voltage clamped connexin32-transfected SKHep1 cells, macroscopic gj was greater than 1 nS; gj increased with time after dissociation (from 1.8 +/- 0.6 [mean +/- SE; n = 7] nS at 2 h after plating to 9.3 +/- 2.2 [n = 9] nS, the maximal value, at 24 h). Unitary conductance of gap junction channels between pairs of transfected SKHep1 cells was measured in low conductance pairs and after reducing gj by exposure to halothane or heptanol. Histograms of gamma j values in transfected cells, in 10 experiments where greater than 100 transitions were measurable, displayed two peaks; 120-130 pS and 25-35 pS. The smaller size corresponded to channels that were occasionally detected in the parental cells. We therefore conclude that connexin32 forms gap junctions channels of the 120-130 pS size class.     

Further studies on the effect of intracellular angiotensins on heart cell communication: on the role of endogenous angiotensin II

The influence of intracellular angiotensin I (Ang I) and angiotensin II (Ang II) on the process of cell communication was investigated in isolated cell pairs from the failing heart of cardiomyopathic hamsters at 2 and at 6 months of age. Measurements of junctional conductance were performed on weekly coupled ventricular cells (4-5.3 nS) using two separated voltage clamp circuits. The results indicated that at 2 months of age, when no signs of heart failure are detected, the angiotensin converting enzyme (ACE) activity is low and similar to controls (0.26 nmol/mg/min). Here the intracellular dialysis of angiotensin I (10(-8) M) caused a decline of junctional conductance of 33+/-3.6% (n=35) (P<0.05) within 10 min while the administration of the same concentration of Ang I elicited cell uncoupling in cell pairs of 6-month-old cardiomyopathic hamsters in which the ACE activity was enhanced (0.41+/-0.05 nmol/mg/min) (P<0.05). Intracellular administration of angiotensin II in cell pairs of 2-month-old hamsters caused a decline of junctional conductance of only 25+/-4.5% (n=35) (P<0.05) compared to cell uncoupling in 6-month-old cardiomyopathic hamsters. Intracellular losartan(10(-8) M) reduced the effect of intracellular Ang II by 68+/-3.5% (n=28) on 2-month-old hamsters and abolished the effect of the peptide on 6-month-old hamsters. To investigate the influence of endogenous angiotensin II on the regulation of cell coupling, enalapril maleate (10(-8) M) or enalaprilat (10(-9) M) was used. The results indicated that at 2 months of age, no change in cell coupling was elicited by the ACE inhibitor while at 6 months of age, there was an increment of cell coupling of 72+/-6.2% (P<0.05). Similar results were found with intracellular losartan (10(-8) M). These results support the view that endogenous angiotensin II is involved in the regulation of cell communication at an advanced stage of heart failure when the ACE activity is enhanced and the cardiac renin angiotensin system (RAS) is activated.     

Gap junctional communication of primary human keratinocytes: characterization by dual voltage clamp and dye transfer.

We have compared dye coupling in pairs of small (less than 10 microns in diameter) and large (greater than 20 microns in diameter) keratinocytes isolated from normal human epidermis, using Lucifer yellow microinjection. Under control conditions, dye coupling was found in only 1 out of the 25 small pairs tested, whereas it was evident in 75% of the large pairs (n = 52). After a 30-min incubation of the latter pairs in the presence of 10(-6) and 10(-4) M all-transretinoic acid (RA), the percentage of coupling was 53% (n = 15; NS) and 7% (n = 14; P less than 0.001), respectively. The almost complete uncoupling observed after 10(-4) M RA was not reversible even 30 min after return to control medium (n = 8). Dual whole-cell patch-clamp recordings from large keratinocyte pairs showed a macroscopic junctional conductance (gj) of 9 +/- 2 nS (n = 43), which was abolished by heptanol (3.5 mM) in a fully reversible way. Compared to heptanol, 10(-4) M RA abolished keratinocyte gj more slowly and irreversibly (n = 10). By contrast, 10(-6) M RA had no significant effect on gj (n = 8). Single-gap junctional channels were also identified between large keratinocytes. Events histograms of 152 transitions from three experiments revealed three main unitary conductances (gamma j) of 45 +/- 4, 78 +/- 4, and 106 +/- 7 pS. The dye coupling results indicate that junctional communication is markedly different in pairs of small and large cells, which showed the phenotype and keratin markers of basal and suprabasal keratinocytes, respectively. In the latter cell type, coupling is ensured by channels of three sizes and is blocked irreversibly by pharmacologic concentrations of RA.     

Single-membrane and cell-to-cell permeability properties of dissociated embryonic chick lens cells

Ion channels are believed to play an important role in the maintenance of lens transparency. In order to ascribe junctional and nonjunctional permeability properties to specific lens cell types, embryonic chick lenses were enzymatically dissociated into cell clusters, cell pairs and single cells, and both cell-to-cell and single-membrane permeability properties were characterized with the patch-clamp technique. Double patch-clamp experiments and single patch-clamp experiments with Lucifer yellow in the pipette demonstrated that the cells in the dissociated preparation were well coupled, the average conductance between pairs being 42 +/- 27 nS. Double patch-clamp experiments also revealed single cell-to-cell channel events with a predominant unitary conductance of 286 +/- 38 pS. Whole-cell measurements of surface membrane conductance indicate heterogeneity within the population of dissociated embryonic chick lens cells: 63% of the cells have a voltage-independent leak current, 14% of the cells have a potassium-selective inward-rectifier current, and 23% of the cells have a current which turns off with positive voltage on a time scale on the order of seconds. The time constant for this turnoff is voltage dependent.     

Voltage clamp limitations of dual whole-cell gap junction current and voltage recordings. I. Conductance measurements

Previous correction methods for series access resistance errors in the dual whole-cell configuration did not take into account the effect of nonzero resting potentials (E(rest)) and junctional reversal potentials (E(rev)). Dual whole-cell currents were modeled according to resistor-circuit analysis and two correction formulas for the measurement of junctional currents (I(j)) were assessed. The equations for I(j), derived from Kirchoff's law before and after baseline subtraction of the nonjunctional current, were assessed for accuracy under a variety of whole-cell patch-clamp recording conditions. Both equations accurately correct for dual whole-cell voltage-clamp errors provided that the cellular parameters are included in the nonbaseline subtracted I(j) derivations. Junctional conductance (g(j)) estimates are most reliable at high junctional resistance (R(j)) values and minimize the need for corrective methods based on electrode series and cellular input resistances (R(el) and R(in)). In the "open-cell" configuration, low R(j) values relative to R(in) are required for accurate g(j) estimates. These methods provide the basis for accurate quantitative measurements of junctional resistance (or conductance) of gap junction channels or connexin hemichannels in the dual whole-cell or open-cell configurations. Revaluation of V(j)-dependent gating of rat connexin40 g(j) produced nearly identical Boltzmann fits to previously published data. Continuous g(j)-V(j) curves generated by variable slope V(j) ramps provide for more accurate fits and assessment of the time-dependence of the half-inactivation voltage and net gating charge movement.     

Voltage-clamp studies of gap junctions between uterine muscle cells during term and preterm labor.

Gap junctions between myometrial cells increase dramatically during the final stages of pregnancy. To study the functional consequences, we have applied the double-whole-cell voltage-clamp technique to freshly isolated pairs of cells from rat circular and longitudinal myometrium. Junctional conductance was greater between circular muscle-cell pairs from rats delivering either at term (32 +/- 16 nS, mean +/- SD, n = 128) or preterm (26 +/- 17 nS, n = 33) compared with normal preterm (4.7 +/- 7.6 nS, n = 114) and postpartum (6.5 +/- 10 nS, n = 16); cell pairs from the longitudinal layer showed similar differences. The macroscopic gap junction currents decayed slowly from an instantaneous, constant-conductance level to a steady-state level described by quasisymmetrical Boltzmann functions of transjunctional voltage. In half of circular-layer cell pairs, the voltage dependence of myometrial gap junction conductance is more apparent at smaller transjunctional voltages (< 30 mV) than for other tissues expressing mainly connexin-43. This unusual degree of voltage dependence, although slow, operates over time intervals that are physiologically relevant for uterine muscle. Using weakly coupled pairs, we observed two unitary conductance states: 85 pS (85-90% of events) and 25 pS. These measurements of junctional conductance support the hypothesis that heightened electrical coupling between the smooth muscle cells of the uterine wall emerges late in pregnancy, in preparation for the massive, coordinate contractions of labor.     

Effects of transmembrane gradients of bicarbonate and ammonium on junctional and nonjunctional membrane conductances in BHK cells

Weak acids are efficient blockers of gap-junctional conductance. It is generally accepted that intracellular acidification produced by weak acids fully accounts for the gap-junctional uncoupling. Protonation of the cytoplasmic portions of the channel-forming protein connexin is thought to lead to the conformational changes switching the channel from the open into the closed state. If this is the only mechanism of the weak-acid induced uncoupling, then the correlation between junctional conductance (Gj) and intracellular pH (pHin) should not depend on the means of intracellular acidification. We compared the responses of junctional conductance in BHK cells measured in double whole-cell experiments to the applied transmembrane concentration gradients of bicarbonate or ammonium. These treatments were to lower pHin in a predictable way according to the equations: pHin = pHout -lg[[HCO3]out/HCO3-]in) or pHin = PHout - lg[[NH4+]in[NH4+out), respectively. We found that the behavior of Gj depended on the substance used. At a 500-fold bicarbonate gradient (calculated pHin approximately 4.8) the cells remained coupled, while a 100- or 10-fold gradient of ammonium imposing pHin approximately 6.1 produced fast uncoupling. The responses of junctional conductance were often accompanied or preceded by changes of non-junctional membrane conductance. We suggest that the mechanisms of the weak acid/base-induced channel gating may contain an additional "lipophilic" component due to the presence of the non-dissociated form of the acid/base in cell membrane.     

乙酰胆碱对蟾蜍背根神经节神经元膜电位的影响及离子机制

在离体灌流的蟾蜍背根神经节(DRG)标本上,用微电极进行胞内记录。在73个神经元中,依神经纤维的传导速度将神经元分为 A 型及 C 型,其中 A 型细胞67个,C 型6个,静息膜电位为-67.5±1.3mV((?)±SE)。当加4×10~(-4)—6×10~(-4)mol/L 乙酰胆碱(ACh),可观察到如下四种膜电位变化:1.超极化:幅值9.1±3.0mV((?)±SE,n=23);(2)去极化:幅值12.9±2.2mV((?)+SE,n=20);(3)双相反应(n=24):先超极化,后去极化,超极化幅值8.0±2.4mV((?)+SE),去极化幅值10.9±3.1mV((?)±SE);(4)无反应(n=6)。用阿托品(1.3×10~(-5)mol/L,n=23),或同时应用筒箭毒与六甲双铵(浓度均为1.4×10~(-5)mol/L,n=8)灌流,能分别阻断 ACh 引起的膜的超极化或去极化。ACh 引起超极化反应时膜电导平均增加13.8%,翻转电位值大约-96mV。四乙铵(TEA,20mmol/L)能使 ACh 的去极化幅值增加48.2±3.2%((?)±SE,n=6),超极化幅值减小79.4±4.3%((?)±SE,n=8)。MnCl_2(4mmol/L)使 ACh 的去极化及超极化幅值分别减小54.2±7.2%((?)±SE,n=5)及69.2±6.4%((?)±SE,n=14)。以上结果提示:ACh 引起的 DRG 神经细胞膜去极化反应由 N 型乙酰胆碱受体介导,而超极化反应由 Μ 型乙酰胆碱受体介导,前者可能包含了多种离子电导的改变,后者则可能与钾电导增加有关。     

Gap-junctional single-channel permeability for fluorescent tracers in mammalian cell cultures

We have developed a simple dye transfer method that allows quantification of the gap-junction permeability of small cultured cells. Fluorescent dyes (calcein and Lucifer yellow) were perfused into one cell of an isolated cell pair using a patch-type micropipette in the tight-seal whole cell configuration. Dye spreading into the neighboring cells was monitored using a low-light charge-coupled device camera. Permeation rates for calcein and Lucifer yellow were then estimated by fitting the time course of the fluorescence intensities in both cells. For curve fitting, we used a set of model equations derived from a compartment model of dye distribution. The permeation rates were correlated to the total ionic conductance of the gap junction measured immediately after the perfusion experiment. Assuming that dye permeation is through a unit-conductance channel, we were then able to calculate the single-channel permeance for each tracer dye. We have applied this technique to HeLa cells stably transfected with rat-Cx46 and Cx43, and to BICR/M1R(k) cells, a rat mammary tumor cell line that has very high dye coupling through endogenous Cx43 channels. Scatter plots of permeation rates versus junctional conductance did not show a strictly linear correlation of ionic versus dye permeance, as would have been expected for a simple pore. Instead, we found that the data scatter within a wide range of different single-channel permeances. In BICR/M1R(k) cells, the lower limiting single-channel permeance is 2.2 +/- 2.0 x 10(-12) mm3/s and the upper limit is 50 x 10(-12) mm3/s for calcein and 6.8 +/- 2.8 x 10(-12) mm3/s and 150 x 10(-12) mm3/s for Lucifer yellow, respectively. In HeLa-Cx43 transfectants we found 2.0 +/- 2.4 x 10(-12) mm3/s and 95 x 10(-12) mm3/s for calcein and 2.1 +/- 6.8 x 10(-12) mm3/s and 80 x 10(-12) mm3/s for Lucifer yellow, and in HeLa-Cx46 transfectants 1.7 +/- 0.3 x 10(-12) mm3/s and 120 x 10(-12) mm3/s for calcein and 1.3 +/- 1.1 x 10(-12) mm3/s and 34 x 10(-12) mm3/s for Lucifer yellow, respectively. This variability is most likely due to a yet unknown mechanism that differentially regulates single-channel permeability for larger molecules and for small inorganic ions.     

Mapping and quantifying hyperpolarized 3He magnetic resonance imaging apparent diffusion coefficient gradients.

We measured hyperpolarized 3He magnetic resonance imaging (MRI) apparent diffusion coefficients (ADC) and quantified ADC gradients in each three-by-three voxel region of interest (ROI). Such local ADC gradients can be represented in vector maps showing the magnitude (|G3x3|) and direction of ADC gradients, providing a qualitative visualization tool and quantitative measurement of airway and air space heterogeneity. Twenty-four subjects (15 male, mean age=67+/-7 yr) with global initiative for chronic obstructive lung disease (GOLD) stage II (n=9, mean age 68+/-6 yr), GOLD stage III chronic obstructive pulmonary disease (COPD; n=7, mean age 67+/-8 yr), and age-matched healthy volunteers (n=8, mean age 67+/-6 yr) were enrolled based on their age and spirometry results. Hyperpolarized 3He MRI was performed on a whole body 3.0 Tesla system. Mean 3He ADC and ADC standard deviation were calculated for the center coronal slice, and the mean magnitude and direction of the ADC gradient vectors were calculated for each three-by-three voxel matrix (|G3x3|). While the 3He ADC standard deviation was not significantly different, mean |G3x3| was significantly different between subjects with stage II (0.14+/-0.03 cm/s) and stage III COPD (0.19+/-0.03 cm/s; P<0.005) and between healthy subjects (0.12+/-0.03 cm/s) and those with stage II COPD (P<0.02). The second order statistic |G3x3| may provide a sensitive measure of ADC heterogeneity for ROI representing 9.4x9.4x30 mm or 2.6 cm3 of lung tissue.     

An insulin-stimulated cation channel in skeletal muscle. Inhibition by calcium causes oscillation.

A cation channel has been identified in the plasma membrane of skeletal muscle that oscillates open and closed in a regular manner. In an experimental system of patch-clamped reconstituted plasma membrane in phospholipid bilayers, the oscillations are calcium-dependent and constitute regular closing events due to inhibition of the channel by calcium with a Ki of 2.2 +/- 1 x 10(-6) M, followed by reopening. There are 3.7 +/- 1 calcium binding sites/channel. With sodium as the current vehicle, conductance is increased by voltage, insulin (Km = 5 +/- 0.6 x 10(-9) M), and hydrolyzable guanine nucleotides. Cyclic GMP alone with increase the conductance with a Km of 3.7 +/- 0.6 x 10(-7) M. In the absence of calcium, the unitary conductance with insulin + GTP or cGMP at 150 mM NaCl is 153 picosiemens. Sodium current is insensitive to 10(-5) M tetrodotoxin but inhibited by mu-conotoxin (Ki = 5 x 10(-8) M). These findings in the reconstituted system were verified in patch-clamped whole muscle cells where an insulin and cGMP-dependent sodium current inhibited by mu-conotoxin could be demonstrated. In the whole cell experiments, slow calcium-dependent oscillations of the sodium current were also detected.     

Junctional transfer in cultured vascular endothelium: I. Electrical coupling

Vascular endothelial cultures are composed of flat, polygonal monolayer cells which retain many of the growth, metabolic and physiological characteristics of the intimal endothelium. However, intercellular gap and tight junctions, which are thought to perform important roles in normal intimal physiology, are reduced in complexity and extent in culture. We have used electrophysiological techniques to test confluent (3- to 5-day) primary cultures of calf aortic (BAEC) and umbilical cord vein (BVEC) endothelium for junctional transfer of small ions. Both cell types are extensively electrically coupled. The passive electrical properties of the cultured cells were calculated from the decrease in induced membrane potential deflections with distance from an intracellular, hyperpolarizing electrode. Data analyses were based on a thin-sheet model for current flow (Bessel function). The generalized space constants (lambda) were 208.6 microns (BAEC) and 288.9 microns (BVEC). The nonjunctional (6.14 and 8.72 X 10(8) omega) and junctional (3.67 and 3.60 X 10(6) omega) resistances were similar for the BAEC and BVEC, respectively. We detected no statistically significant differences in the resistance estimates for the two cell types. In vivo ultrastructural studies have suggested that aortic endothelium has more extensive gap junctions than venous endothelium. We have found that these ultrastructural differences are reduced in culture. The lack of any significant difference in electrical coupling capability suggests that cultured BAEC and BVEC have functionally similar junctional characteristics.     

Dog liver glucose-6-phosphate dehydrogenase: purification and kinetic properties

Glucose-6-phosphate dehydrogenase (G6PD) catalyses the first step of the pentose phosphate pathway which generates NADPH for anabolic pathways and protection systems in liver. G6PD was purified from dog liver with a specific activity of 130 U x mg(-1) and a yield of 18%. PAGE showed two bands on protein staining; only the slower moving band had G6PD activity. The observation of one band on SDS/PAGE with M(r) of 52.5 kDa suggested the faster moving band on native protein staining was the monomeric form of the enzyme.Dog liver G6PD had a pH optimum of 7.8. The activation energy, activation enthalpy, and Q10, for the enzymatic reaction were calculated to be 8.96, 8.34 kcal x mol(-1), and 1.62, respectively.The enzyme obeyed "Rapid Equilibrium Random Bi Bi" kinetic model with Km values of 122 +/- 18 microM for glucose-6-phosphate (G6P) and 10 +/- 1 microM for NADP. G6P and 2-deoxyglucose-6-phosphate were used with catalytic efficiencies (kcat/Km) of 1.86 x 10(6) and 5.55 x 10(6) M(-1) x s(-1), respectively. The intrinsic Km value for 2-deoxyglucose-6-phosphate was 24 +/- 4mM. Deamino-NADP (d-NADP) could replace NADP as coenzyme. With G6P as cosubstrate, Km d-ANADP was 23 +/- 3mM; Km for G6P remained the same as with NADP as coenzyme (122 +/- 18 microM). The catalytic efficiencies of NADP and d-ANADP (G6P as substrate) were 2.28 x 10(7) and 6.76 x 10(6) M(-1) x s(-1), respectively. Dog liver G6PD was inhibited competitively by NADPH (K(i)=12.0 +/- 7.0 microM). Low K(i) indicates tight enzyme:NADPH binding and the importance of NADPH in the regulation of the pentose phosphate pathway.     

Stimulation of secretion into human and feline airways by Pseudomonas aeruginosa proteases

We have investigated the effect of elastase and alkaline protease from Pseudomonas aeruginosa on airway secretion into the trachea of anesthetized cats and from human bronchial mucosa in vitro. Secretory macromolecules were radiolabeled biosynthetically with two precursors in the cat, [3H]glucose and [35S]sulfate, and with [35S]-sulfate only in human tissue. Both enzymes (2.6 x 10(-9) to 1.3 x 10(-6)M elastase and 8 x 10(-9) to 2.4 x 10(-6)M alkaline protease) released radiolabeled macromolecules in a concentration-dependent manner from the two preparations. Purified elastase, 1.3 x 10(-6)M, released radiolabeled macromolecules (delta 3H = +397 +/- 72%, delta 35S 225 +/- 40% over control, P less than 0.001) and periodic acid-Schiff- (PAS) reactive glycoconjugates (delta PAS = +4.1 +/- 0.96 micrograms/min or +102 +/- 20%; P less than 0.01) from cat trachea, as did alkaline protease, 2.4 x 10(-6)M (delta 3H = +356 +/- 57%, delta 35S = +176 +/- 25%, delta PAS = +7.5 +/- 1.3 micrograms/min or 194 +/- 36%, P less than 0.001). Increases in 3H exceeded those of 35S, suggesting surface epithelium as the main source of secretion. Inhibition of enzyme activity abolished secretory effects. Both enzymes also stimulated secretion from human bronchus (e.g., with elastase, 1.3 x 10(-6)M: delta 35S = +331 +/- 67%, delta PAS = +4.3 +/- 0.92 micrograms/min or +131 +/- 24%, P less than 0.001; with alkaline protease, 2.4 x 10(-6)M: delta 35S = +220 +/- 67%, delta PAS = +12.7 +/- 3.2 micrograms/min or +575 +/- 245%, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Opening hemichannels in nonjunctional membrane stimulates gap junction formation

We studied gap junction formation in pairs of Xenopus laevis oocytes expressing connexins that form functional hemichannels and found no correlation between junctional conductance (G(j)) and whole-cell hemichannel conductances (G(hemi)) within the first few hours of pairing. However, opening hemichannels to a threshold current stimulated a rapid G(j) increase. Moreover, cx46 hemichannel current stimulated cx40 G(j) even though cx40 and cx46 do not form heteromeric or heterotypic gap junctions. Initial growth rate and final steady-state level of stimulated G(j) were proportional to the product of hemichannel conductances. External calcium affected the growth rate of stimulated G(j) but not the final steady-state value. Time constants of formation were short in low [Ca(2+)](out) (3 min in 200 micro M Ca(2+)) and long in high [Ca(2+)](out) (15 min in 1 mM Ca(2+)), but in oocyte pairs pretreated with lectins to reduce steric hindrance imposed by large membrane glycoproteins the time constant was short and Ca(2+)-independent. We suggest that hemichannel activity stimulates G(j) by collapsing the extracellular volume between membranes to allow the end-to-end binding between hemichannels. These studies suggest the possibility that functional hemichannels could trigger or enhance junctional formation in vivo in response to appropriate stimuli.     

A quantitative histological study of the aortic nerve and the aortic body in the buffalo (Bubalus bubalis)

In the buffalo, the left aortic nerve ramifies in the periarterial connective tissue between the ventral surface of the aortic arch and the truncus pulmonalis. The right aortic nerve ramifies over the dorsal and right aspects of the aorta ascendens near its origin. The histograms of myelinated fibres of both left and right aortic nerve are distinctly unimodal with peak around 4-6 micron (64.2-67.8%). The left aortic body is situated in the periarterial connective tissue between the ventral surface of the aortic arch and the truncus pulmonalis, while the right aortic body is located in the tunica adventitia of the dorsal and right aspects of the aorta ascendens near its origin. The greatest sagittal section area of the left aortic body is 0.102 +/- 0.009 mm2 and that of the right aortic body is 0.041 +/- 0.002 mm2. The organ is highly vascular. The mean size of the glomus cells from the left aortic body is 7.68 +/- 0.9 micron x 9.37 +/- 0.13 micron (short diameter x long diameter), whereas the corresponding value for the right aortic body is 7.84 +/- 0.14 micron x 9.86 +/- 0.21 micron; and their density values are (11,417 +/- 301.7)/mm2 and (9,839 +/- 213.3)/mm2 respectively.