Glycerol permeability of mutant aquaporin 1 and other AQP-MIP proteins: Inhibition studies

In a recent work, we showed that the aquaporins 1 (AQP1) are permeable to certain small solutes such as glycerol. Here, we have further investigated the permeation pathway of glycerol through human AQP1 (hAQP1) by the use of mutants (C189S, H180A, H209A) and inhibitors such asP-chloromercuribenzene sulphonate (pCMBS), CuSO₄ or phloretin, in comparison with other AQP-MIP (where MIP denotes major intrinsic protein) proteins: hAQP2, plant water channel TIP and bacterial glycerol permease facilitator, G1pF. Glycerol movements were measured inXenopus laevis oocytes. Apparent glycerol permeability coefficients (P _gly ) were calculated from the rates of oocyte swelling upon exposure to an isoosmotic medium containing an inwardly directed gradient of glycerol and from [³H]glycerol uptake measurements. SimilarP _gly values were obtained for hAQP1 and hAQP2, 6 to 8 times greater than control indicating that hAQP2 also transports glycerol. Pof hAQP2P injected oocytes waspCMBS and CuSO₄ sensitive. In contrast, theP _gly value of TIP was close to that of control, indicating that TIP does not transport glycerol. The hAQP1-C189S, -H180A and -H209A mutants gaveP _gly values similar to those obtained for wild hAQP1, indicating that these mutations did not affect glycerol movements. However, the H209A mutant has an osmotic water permeability coefficient (P _f) value decreased by 50%. The inhibitory effect ofpCMBS onP _gly was maintained for the 2 His mutants and, more interestingly, was also conserved for the C189S mutant. CuSO₄ significantly inhibitedP _gly of oocytes expressing hAQP1, hAQP1-C189S,-H180A, and -H209A mutants and had no effect onP _gly of G1pF-injected oocytes. Phloretin was shown to inhibit by around 80% the glycerol fluxes of wild and mutant hAQP1, hAQP2 and to fully inhibit glycerol uptake in G1pF-injected oocytes.     

Modulation of L-type Ca channel activity by P2-purinergic agonist in cardiac cells

The mechanism of enhancement of the L-type Ca current by a P₂-purinergic agonist adenosine-5-O-(3-thiotriphosphate) (ATPS) was studied by recording single channel activity from cell-attached patches on rat isolated ventricular cells using patch pipettes containing 110 mM Ba²⁺. The application of ATPS to the patch membrane through the pipette solution did not affect single channel activity. The addition of ATPS to the bath containing a depolarizing solution was ineffective due to the voltage dependence of the purinergic stimulation. Bath application of ATPS (100 M) to control 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES) solution increased the amplitude of ensemble average currents both by decreasing the probability of a blank sweep occurring and by increasing the number of openings per non-blank sweep. The single channel conductance (17 pS) was not changed by ATPS. Both activation and inactivation curves were shifted towards hyperpolarized potentials by about 10 mV under P₂-purinergic stimulation. Since ATPS increased channel activity when applied via the bath, it must be supposed that a diffusible messenger is involved.     

Absence of long-term modulation of ventilation by dead-space loading during moderate exercise in humans

The stability of arterial PCO₂ (P_aCO₂) during moderate exercise in humans suggests a CO₂-linked control that matches ventilation (_E) to pulmonary CO₂ clearance (CO₂). An alternative view is that _E is subject to long-term modulation (LTM) induced by hyperpnoeic history. LTM has been reported with associative conditioning via dead-space (V_D) loading in exercising goats (Martin and Mitchell 1993). Whether this prevails in humans is less clear, which may reflect differences in study design (e.g. subject familiarisation; V_D load; whether or not _E is expressed relative to CO₂; choice of P_aCO₂ estimator). After familiarisation, nine healthy males performed moderate constant-load cycle-ergometry (20 W-80 W-20 W; <lactate threshold, _L): day 1, pre-conditioning, n=3; day 2, conditioning (V_D=1.59 l, doubling _E at 20 W and 80 W), n=8 with 10 min rest between tests; and, after 1 h rest, post-conditioning, n=3. Gas exchange was determined breath-by-breath. Post-conditioning, neither the transient [phase 1, phase 2 (1, 2)] nor steady-state _E exercise responses, nor their proportionality to CO₂, differed from pre-conditioning. For post-conditioning trial 1, steady-state _E was 28.1 (4.7) l min^–1 versus 29.1 (3.8) l min^–1 pre-conditioning, and mean-alveolar PCO₂ (a validated P_aCO₂ estimator) was 5.53 (0.48) kPa [41.5 (3.6) mmHg] versus 5.59 (0.49) kPa [41.9 (3.7) mmHg]; the 1 _E increment was 4.2 (2.9) l min^–1 versus 5.2 (1.9) l min^–1; the 2 _E time-constant () was 64.4 (24.1) s versus 64.1 (25.3) s; _E/CO₂ was 1.12 (0.04) versus 1.10 (0.04); and the _E-CO₂ slope was 21.7 (3.4) versus 21.2 (3.2). In conclusion, we could find no evidence to support ventilatory control during moderate exercise being influenced by hyperpnoeic history associated with dead-space loading in humans.     

ω-Grammotoxin blocks action-potential-induced Ca2+ influx and whole-cell Ca2+ current in rat dorsal-root ganglion neurons

Field-potential stimulation of rat dorsal-root ganglion (DRG) neurons evoked action-potential-mediated transient increases in intracellular free calcium concentration ([Ca²⁺]_i) as measured by indo-1-based microfluorimetry. Field-potential-evoked [Ca²⁺]_i transients were abolished by tetrodotoxin, and their dependence on stimulus intensity exhibited an abrupt threshold. -Conotoxin GVIA (-CgTx, 100 nM) inhibited action-potential-mediated Ca²⁺ influx by 79%, while nitrendipine (1 M) had little effect. -Grammotoxin SIA (-GsTx, 267 nM), a peptide toxin purified from the venom of the tarantula spider, Grammostola spatulata, blocked action-potential-mediated Ca²⁺ influx as effectively as did -CgTx, suggesting that -GsTx blocks N-type Ca²⁺ channels. In contrast to block by -CgTx, the block produced by -GsTx reversed upon washout of the peptide. -GsTx (270 nM) blocked 80%, and -CgTx (1 M) blocked 64%, of whole-cell Ca²⁺ current (I _Ca) elicited by step depolarization to 0 mV from a holding potential of –80 mV. -GsTx completely occluded inhibition of I _Ca by -CgTx. However, when applied after -CgTx, -GsTx produced an additional inhibition of 27%, indicating that -GsTx also blocked a non-N-type Ca²⁺ channel. BayK8644 (1 M) elicited an increase in I _Ca in the presence of maximally effective concentrations of -GsTx, suggesting that -GsTx does not block L-type channels. Thus, -GsTx displays a selectivity for Ca²⁺ channel subtypes which should prove useful for studying Ca²⁺ channels and Ca²⁺-channel-mediated processes.     

An in vitro model to study myocardial ischemic injury

Summary This report describes a method to study ischemic and reperfusion damage in cultured ventricular cardiomyocytes. The cardiomyocytes were made ischemic by oxygen deprivation and volume restriction. The ischemic intervals studied were 60, 90, and 120 min. An oxygen tension of 0 mmHg was maintained throughout ischemia. Reperfusion was simulated by bathing the cells in a large volume of normally oxygenated PBSG following ischemia. Ischemia and reperfusion resulted in increased lactate production, increased membrane damage, and ATP degradation. Ischemia caused a decrease in pH. Although 90 min of ischemia caused cell structural changes and alteration of metabolism, a prolonged ischemic interval of 120 min exacerbated these abnormalities. We have found our in vitro ischemia and reperfusion model to closely mimic the in vivo condition. This model using human ventricular cardiomyocytes is a means to studying the effect and mechanism of ischemia and reperfusion at the cellular level under defined conditions.     

Disinhibition of hippocampal pyramidal cells during the transition into theta rhythm

The activity of hippocampal complex-spike cells (presumed pyramidal cells) and theta cells (presumed interneurons) was examined during transitions from non-theta electroencephalogram (EEG) states to theta EEG states in freely moving and sleeping rats. Theta cell firing rates were significantly depressed in a 1-s period centered on the EEG transition relative to the surrounding 1-s periods (normalized rates±SEM): 1.05±0.02 for the non-theta period, 0.59±0.03 for the transition period, and 1.36±0.04 for the theta period (n = 26 cells). Conversely, complex-spike cell firing was significantly increased during the transition period: 0.51±0.11 for the non-theta period, 2.24±0.19 for the transition period, and 0.24±0.04 for the theta period (n = 27 cells). This diametrically altered activity indicates that theta cells must be actively inhibited during the transition. The increased activity in complex-spike cells during the transition may be simply a release from inhibitory control by interneurons. The pattern of theta cell inhibition together with increased complex-spike cell activity appears to be a general property of transitions into the theta EEG state, irrespective of behavior. It is suggested that increased activity in septal afferents (GABAergic cell activity greater than cholinergic cell activity) initially inhibits hippocampal interneurons. The inhibition is not sustained because of an activity-dependent decrease in the potency of the septointerneuronal inhibition, leaving the rhythmic excitatory (cholinergic) septointerneuronal inputs, together with principal cell inputs, to increase interneuron firing rates.     

Effect of posture on arterial baroreflex control of heart rate in humans

Summary Altered baroreflex function may contribute to the cardiovascular changes associated with weightlessness. Since central blood volume (CBV) increases during simulated weightlessness, we have examined the possibility that acute changes in CBV may modify baroreceptor function. We used graded head-up tilt (HUT) and head-down tilt (HDT) to induce changes in CBV, and neck suction to stimulte carotid baroreceptors, in 6 subjects. The increase in pulse interval induced by a negative pressure of 8.2 kPa (62 mm Hg) imposed for 10 s while supine was compared with the increase while tilted for 8 min at ± 15, ± 30 and ± 45. During HDT at 15 the pulse interval over the first 5 cardiac cycles following suction onset was 51 ± (SEM) 18 ms longer (p<0.05), at 30 it was 61±20 ms longer (p<0.05), and at 45 it was 74±35 ms longer (p<0.01), compared with supine. During HUT at 15 the pulse interval was 25±9 ms shorter (p<0.05) than when supine, but was not significantly different at 30 and 45. These responses occurred independently of changes in brachial blood pressure. Attenuation was also observed after 5 min (56±17 ms; <0.05), and after 40 min (25±9 ms; p<0.05) of 60 HUT compared with supine. We conclude that posture does modify arterial baroreflex control of heart rate. If this occurs primarily as a result of a change in CBV, then the acute effect of weightlessness may be an accentuation, not an attenuation, of baroreflex function.M. H. Harrison was a National Research Council postdoctoral research fellow on leave from the Ministry of Defence, UK     

α 2-Adrenoceptors activate dihydropyridine-sensitive calcium channels via Gi-proteins and protein kinase C in rat portal vein myocytes

The presence of functional ₂-adrenoceptors was investigated in isolated smooth muscle cells from rat portal vein using the nystatin-perforated patch-clamp technique. The free cytoplasmic calcium concentration ([Ca²⁺]_i) was estimated using emission from the dye Fura-2. Activation of ₂-adrenoceptors by clonidine (an ₂-adrenoceptor agonist) or noradrenaline (a non-selective -adrenoceptor agonist), both in the presence of 0.1 M prazosin to block ₁-adrenoceptors, caused a slow and sustained increase in [Ca²⁺]_i which was inhibited by 0.1 M rauwolscine (an ₂-adrenoceptor antagonist). A similar Ca²⁺ response was obtained with oxymetazoline (a selective _2A-adrenoceptor agonist) suggesting that the increase in [Ca²⁺]_i resulted from activation of the _2A-adrenoceptor subtype. The increase in [Ca²⁺]_i did not occur in calcium-free solution or in the presence of oxodipine (a voltage-dependent calcium channel blocker), indicating that it depended on a calcium influx. The _2A-adrenoceptor-activated calcium influx was unchanged after complete release of the stored calcium induced by applications of ryanodine and caffeine. In addition, no accumulation of inositol trisphosphate was detected in the presence of 0.1 M prazosin. Taken together, these results indicate that _2A-adrenoceptor activation does not stimulate phosphoinositide turnover and subsequent calcium release from intracellular stores. Wholecell patch-clamp experiments showed that _2A-adrenoceptor activation promoted calcium influx through voltage-dependent L-type channels. Concomitant with calcium influx, _2A-adrenoceptor activation induced a 10- to 15-mV depolarization. Similar effects on both calcium channel current and [Ca²⁺]_i were obtained with mastoparan, an activator of Gi-proteins. Activation of calcium influx by both _2A-adrenoceptors and mastoparan was reduced by treatment with pertussis toxin and GF 109203X (a protein kinase C inhibitor). These data suggest that activation of protein kinase C through a transduction pathway involving Gi-proteins phosphorylates voltage-activated L-type calcium channels and thus, increases their opening probability.     

Presence of two isoforms of Na,K-ATPase with different pharmacological and immunological properties in the rat kidney

Previous studies have demonstrated the presence of two populations of Na,K-ATPase with distinct kinetic, pharmacological and immunological characteristics along the rabbit nephron, indicating that the proximal segments of the nephron express exclusively the ₁ isoform of the catalytic subunit, whereas the collecting duct expresses an ₃-like isoform. Because pharmacological studies have shown the existence of two populations of Na,K-ATPase with different sensitivities to ouabain in the rat cortical collecting duct, which may result from the presence in the same nephron segment of the two isoforms demonstrated in the different segments of the rabbit nephron, the present study was undertaken to characterize the properties of Na,K-ATPase along the rat nephron. Results indicate that each segment of the rat nephron contains two subpopulations of Na,K-ATPase: a component highly sensitive to ouabain (IC₅₀ 5.10^–6 M) which is recognized by an anti- ₃ antibody and another moiety of lower affinity for ouabain (IC₅₀ 5.10^–4 M) which is recognized by an anti- ₁ antibody. Whether these two subpopulations correspond to different isoforms of the subunit of Na,K-ATPase ( ₁ and ₃-like) remains to be determined.     

Overshoot in <Emphasis Type="Italic">V̇</Emphasis>O<Subscript>2</Subscript> following the onset of moderate-intensity cycle exercise in trained cyclists

We have previously observed that following the onset of moderate intensity cycle ergometry, the pulmonary O₂ uptake (O₂) in trained cyclists often does not increase towards its steady-state value with the typical mono-exponential characteristics; rather, there is a transient overshoot. The purpose of this study was to systematically examine this phenomenon by comparing the O₂responses to two moderate-intensity work rates and one high-intensity work rate in trained and untrained subjects. Following a ramp exercise test to the limit of tolerance for the determination of the gas exchange threshold (GET) and O_2peak, seven trained cyclists [mean (SD); O_2peak 66.6 (2.5) ml·kg^–1·min^–1] and eight sedentary subjects [O_2peak 42.9 (5.1) ml·kg^–1·min^–1] completed six step transitions from baseline cycling to work rates requiring 60% and 80% GET and three step transitions from baseline cycling to a work rate requiring 50% of the difference between GET and O_2peak (50%). O₂ was measured breath-by-breath and modelled using standard techniques. The sedentary subjects did not overshoot the steady-state O₂ at any intensity. At 60% GET, six of the seven cyclists overshot the steady-state O₂ [by an integral volume of 164 (44) ml between ~45 and 125 s]. At 80% GET, four of the seven cyclists overshot the steady-state O₂ [by an integral volume of 185 (92) ml between ~55 and 140 s]. None of the cyclists showed an overshoot at 50%. These results indicate that trained cyclists evidence an overshoot in O₂ before steady-state is reached in the transition to moderate-intensity exercise. The mechanism(s) responsible for this effect remains to be elucidated, as does whether the overshoot confers any functional or performance benefit to the trained cyclist.     

Characteristics of ecto-ATPase of Xenopus oocytes and the inhibitory actions of suramin on ATP breakdown

Ecto-ATPase activity of Xenopus oocytes was studied by measuring the production of inorganic phosphate (P_i) from the breakdown of extracellular ATP. Enzyme activity involved Ca²⁺/Mg²⁺-dependent and Ca²⁺/Mg²⁺-independent dephosphorylation of ATP. Ca²⁺/Mg²⁺-dependent ecto-ATPase was active over a limited range of 0.01–1.0 mM ATP, while Ca²⁺/Mg²⁺-independent ATPase activity was active over a range of 0.1–30 mM ATP. Total enzyme activity was insensitive to changes in buffer pH (pH 7.0–9.0), but increased in a relatively linear manner with: (1) time of reaction (0–90 min), (2) number of cells (1–20 oocytes), and (3) temperature (10–37°C). Ecto-ATPase activity was unaffected by ouabain (100 M), sodium azide (100 M), and oligomycin (5 g/ml) (as inhibitors of endo-ATPases) and -glycerophosphate (10 mM) and p-nitrophenyl phosphate (10 mM) (as inhibitors of non-specific alkaline phosphatase). Total ecto-ATPase activity was reduced significantly in defolliculated oocytes, suggesting that the enzyme was located mainly on the enveloping follicle cell layer. The range order of preferential substrates was: ATP>GTP, ITP, UTP, CTP, TTP, 2-methylthioATP>ADP, 2-methylthioADP, AMP,-methylene ATP, ,-methylene ATP, in the presence of divalent ions (where G is guanosine, I is inosine, U is uridine, C is cytidine and T is ribosylthymine). The P₂-purinoceptor antagonist suramin [8-(3-benzamido-4-methylbenzamido) napthalene-1,3,5-trisulphonic acid), 100 M] significantly inhibited total ecto-ATPase activity; this inhibition was competitive for the Ca²⁺/Mg²⁺-dependent enzyme. This striking property of suramin may point to a structural similarity between the ATP-binding sites of ecto-ATPase and purinoceptors, a potentially complicating factor where purinoceptors expressed in oocytes are used to test the potency of agonists and the efficacy of receptor antagonists and enzyme inhibitors.     

Go transduces GABAB-receptor modulation of N-type calcium channels in cultured dorsal root ganglion neurons

High-voltage-activated (HVA) calcium channel currents (I _Ba) were recorded from acutely replated cultured dorsal root ganglion (DRG) neurons. I _Ba was irreversibly inhibited by 56.9±2.7% by 1 M -conotoxin-GVIA (-CTx-GVIA), whereas the 1,4-dihydropyridine antagonist nicardipine was ineffective. The selective -aminobutyric acid_B (GABA_B) agonist, (–)-baclofen (50 M), inhibited the HVA I _Ba by 30.7±5.4%. Prior application of -CTx-GVIA completely occluded inhibition of the HVA I _Ba by (–)-baclofen, indicating that in this preparation (–)-baclofen inhibits N-type current. To investigate which G protein subtype was involved, cells were replated in the presence of anti-G protein antisera. Under these conditions the antibodies were shown to enter the cells through transient pores created during the replating procedure. Replating DRGs in the presence of anti-G_o antiserum, raised against the C-terminal decapeptide of the G _o subunit, reduced (–)-baclofen inhibition of the HVA I _Ba, whereas replating DRGs in the presence of the anti-G_i antiseram did not. Using anti-G _o antisera (12000) and confocal laser microscopy, G _o localisation was investigated in both unreplated and replated neurons. G _o immunoreactivity was observed at the plasma membrane, neurites, attachment plaques and perinuclear region, and was particularly pronounced at points of cell-to-cell contact. The plasma membrane G _o immunoreactivity was completely blocked by preincubation with the immunising G_o undecapeptide (1 g · ml^–1) for 1 h at 37° C. A similar treatment also blocked recognition of G _o in brain membranes on immunoblots. These results provide evidence that GABA_B inhibition of N-type calcium channels in acutely replated DRGs occurs via G _o.     

A mutation affecting sexual agglutinability in MATα locus of Saccharomyces cerevisiae

Summary A temperature-sensitive non-agglutinative mutant of Saccharomyces cerevisiae was isolated and characterized. The mutation, sag2, affected sexual agglutination, conjugation and production of -mating pheromone at a restrictive temperature, but not the response to -mating pheromone. Genetic analyses showed that the mutation was recessive and in the MAT locus. The sag2 mutation complemented with mat2 but not with mat1 These results suggest that sag2 is in the MAT1 gene and that at a restrictive temperature the mutation, sag2, inactivated the MAT1 product, a positive regulator of -mating functions. The sag2 mutation is like mat1-5 in its retention of response to -mating pheromone. However, at 25 °C, sag2 cells were competent to mate, whereas mat1-5 cells were not. Hence, sag2 is regarded as a new allele in the MAT1 gene, which we designate mat1-11.     

β subunits determine the time course of desensitization in rat α3 neuronal nicotinic acetylcholine receptors

Standard two-electrode voltage-clamp techniques were used to investigate some of the pharmacological and functional properties of two types of rat neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes after pairwise injection of 34 or 32 mRNAs. Currents of several A amplitude were elicited by fast application of micromolar concentrations of either acetylcholine (ACh) or 1,1-dimethyl-4-piperazine (DMPP). The activation of either receptor type by DMPP showed cooperativity (Hill coefficient, n1.7) with a half-maximal activation concentration (EC₅₀) of 15–30 M. In 34 receptors, ACh displayed cooperativity (n=1.8) but was less efficacious than DMPP, yet its EC₅₀ was about equal to that of DMPP. Finally, in 32 receptors, ACh was much less efficacious and had a much lower EC₅₀. Desensitization induced by either DMPP or ACh was slow in 34 nicotinic ACh receptors but was rapid and extensive in 32 receptors, causing a significant proportion of the response to wane within the first few seconds of agonist application.     

Expression and ligand-binding function of the integrin α4β1 (VLA-4) on neural-crest-derived tumor cell lines

Human neural-crest-derived tumor cell lines, including three neuroblastomas, an astrocytoma, a glioblastoma, a rhabdomyosarcoma and a melanoma were screened for the expression of the integrin ₄₁ (VLA-4). The neuroblastomas IMR-32 and SK-N-SH, the astrocytoma 131-INI, the glioblastoma Fogerty and the rhabdomyosarcoma TE-671 expressed ₄₁ as determined by cytofluorometry and immunoprecipitation. Another neuroblastoma line, LA-N-1, did not express ₄₁. Analysis of immunoprecipitated ₄₁ showed that the ₄ subunit from the various cell types differed in relative molecular weight (M _r ). The variability in the observed M _r could be accounted for by differences in the levels of N-linked glycosylation. The observed variability in M _r did not appear to affect function since intact cells and solubilized ₄₁ bound to a synthetic peptide identical in sequence to the CS-1 region of the alternatively spliced IIICS domain of fibronectin, a known ₄₁, ligand.     

Functional expression and properties of the human skeletal muscle sodium channel

Full-length deoxyribonucleic acid, complementary (cDNA) constructs encoding the-subunit of the adult human skeletal muscle Na⁺ channel, hSkM1, were prepared. Functional expression was studied by electrophysiological recordings from cRNA-injectedXenopus oocytes and from transiently transfected tsA201 cells. The Na⁺ currents of hSkM1 had abnormally slow inactivation kinetics in oocytes, but relatively normal kinetics when expressed in the mammalian cell line. The inactivation kinetics of Na⁺ currents in oocytes, during a depolarization, were fitted by a weighted sum of two decaying exponentials. The time constant of the fast component was comparable to that of the single component observed in mammalian cells. The block of hSkM1 Na⁺ currents by the extracellular toxins tetrodotoxin (TTX) and -conotoxin (CTX) was measured. The IC₅₀ values were 25 nM (TTX) and 1.2 M (CTX) in oocytes. The potency of TTX is similar to that observed for the rat homolog rSkM1, but the potency of CTX is 22-fold lower in hSkM1, primarily due to a higher rate of toxin dissociation in hSkM1. Single-channel recordings were obtained from outside-out patches of oocytes expressing hSkM1. The single-channel conductance, 24.9 pS, is similar to that observed for rSkM1 expressed in oocytes.     

Kinetic and pharmacological properties of high- and low-threshold calcium channels in primary cultures of rat hippocampal neurons

The kinetic, permeability and pharmacological properties of Ca currents were investigated in primary cultures of rat hippocampal neurons. The low-voltage-activated (LVA) Ca current turned on positive to –60mV and fully inactivated in a voltage-dependent way. This current was depressed by nickel (Ni, 40 M) and amiloride (500 M) and was insensitive to -conotoxin (-CgTx) (4 M) and to the Ca agonist Bay K 8644 (5 M). The high-voltage-activated (HVA) Ca current turned on positive to –40 mV and inactivated slowly and incompletely. This current was much less sensitive than the LVA current to Ni and amiloride but more sensitive to cadmium. CgTx blocked only partially this current (about 50%) in an irreversible way. Bay K 8644 had a clear agonistic action almost exclusively on the -CgTx-resistant HVA current component. The present results suggest that the HVA channels, quite homogeneous for their kinetic properties and sensitivity to holding potentials, can be pharmacologically separated in two classes: (i) -CgTx-sensitive and Bay-K-8644-insensitive (-S/BK-I) and (ii) -CgTx-insensitive and Bay-K-8644-sensitive (-I/BK-S), the latter displaying a stronger Cadependent inactivation.     

β-Subunit expression is required for cAMP-dependent increase of cloned cardiac and vascular calcium channel currents

In contrast to vascular smooth muscle (VSM). cAMP-depehdent phosphorylation increases L-type voltage dependent Ca²⁺-channel (L-VDCC) activity in heart. To investigate whether this difference depends on the tissue-specific ₁-subunit of the L-VDCC or its regulation by other subunits, we used a Xenopus laevis oocyte expression system. Injection of cAMP into oocytes expressing cardiac ₁ or VSM ₁ alone had no effect on L-VDCC activity. However, cAMP increased L-VDCC activity 2-fold in oocytes co-expressing cardiac ₁ or VSM ₁ with the skeletal muscle ß-subunit. These results suggest that the presence of the ß-subunit is required for cAMP-mediated increase of L-VDCC activity and that the characteristics of tissue-specific ß-subunits may explain differential regulation of L-VDCC activity.     

Phenotypic and epistatic grouping of hypo- and hyper-rec mus mutants in Aspergillus

The mutants musK to musS of Aspergillus nidulans are sensitive to methyl-methanesulfonate (MMS) and several of them are meiotic-defective and alter mitotic recombination frequencies. All were found to be cross-sensitive to 4-nitro-quinoline-N-oxide (4-NQO) but unexpectedly none of them was hypersensitive to -rays and few to UV light. Double mus;uvs mutants were constructed to test for interactions with uvs mutations of the four epistatic groups of Aspergillus, UvsF, UvsC, UvsI, and UvsB. All meiotic-defective mus mutations caused some lethal interactions, usually with uvsF. None of them showed epistasis with UvsF or UvsB group mutants and one, musO, may represent a new group. Three mus mutations that affect recombination were assigned to the UvsC group, namely musN and K, and also musL which is recombination-defective and closely resembles uvsC. While uvsC mutants are mutators and lack UV-mutagenesis, most mus mutants had no effects on mutation. Only musR, which appeared epistatic with uvsI, showed reduced UV-reversion frequencies similar to uvsI. The recombination-proficient mus mutants appeared to be epistatic with more than one group, but in several cases sensitivities were slight and overlaps insufficient to obtain corroborating results with MMS and 4-NQO.     

Die optische Rotationsdispersion isolierter Paraproteine

Zusammenfassung Die aus dem optischen Drehungsvermögen abgeleiteten Konstanten elektrophoretisch isolierterA-Paraproteine werden mitgeteilt. Die Dispersionskonstante _c weist keine Unterschiede zwischen den 3 ParaproteingruppenG,A undM auf. Der nach dem Verfahren vonMoffitt undYang ermittelte Parameterb ₀ wurde zu Schätzung des-Helixgehaltes benutzt. Er betrug in den 7 untersuchten Paraproteinen 0. Für den Parameter —a ₀ ergab sich ein Mittelwert von 276,0±35,1. FürG-Paraprotein wurde in früheren Untersuchungen ein solcher von 312,8±20,8, fürM-Paraprotein 217,9±26,7 gefunden. Der Mittelwertsvergleich zeigte Signifikanz der Konstantea ₀ für jede der 3 Paraproteingruppen.a ₀ beschreibt demnach gruppenspezifische Eigenschaften von Paraproteinen. Die für den Wert vona ₀ maßgeblichen strukturellen Voraussetzungen sind kaum bekannt. Sie werden am ehesten die die spezifischen Antigendeterminanten tragenden H-Ketten des Paraproteinmoleküls betreffen.

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Summary The constants of the optical rotatory dispersion of electrophoretically isolatedA-paraproteins are communicated. There is no difference between theG,A andM-paraprotein group with respect to the dispersion constant _c . The parameterb ₀ was measured according toMoffitt andYang. The-Helix-content calculated fromb ₀ of 7A-paraproteins was sero (0).The mean value of the parameter —a ₀ was 276±35,1. In earlier experiments it was found that —a ₀ forG-paraproteins is 312,8±20,8 and forM-paraproteins 217,9±26,7. The parametera ₀ of each group differs significantly from the others; in other words,a ₀ is group specific. The structural implications of these findings are discussed.