Mobilization of intracellular calcium in cultured vascular smooth muscle cells by uridine triphosphate and the calcium ionophore A23187.

The known action of uridine triphosphate (UTP) to contract some types of vascular smooth muscle, and the present finding that it is more potent than adenosine triphosphate in eliciting an increase in cytosolic Ca2+ concentration in aortic smooth muscle, led us to investigate the mode of action of this nucleotide. With this aim, cultured bovine aorta cells were subjected to patch-clamp methodologies under various conditions. Nucleotide-induced variations in cytosolic Ca2+ were monitored by using single channel recordings of the high conductance Ca(2+)-activated K+ (Maxi-K) channel within on-cell patches as a reporter, and whole-cell currents were measured following perforation of the patch. In cells bathed in Na(+)-saline, UTP (> 30 nM) induced an inward current, and both Maxi-K channel activity and unitary current amplitude of the Maxi-K channel transiently increased. Repetitive exposures elicited similar responses when 5 to 10 min wash intervals were allowed between challenges of nucleotide. Oscillations in channel activity, but not oscillation in current amplitude were frequently observed with UTP levels > 0.1 microM. Cells bathed in K+ saline (150 mM) were less sensitive to UTP (approximately 5-fold), and did not show an increase in unitary Maxi-K current amplitude. Since the increase in amplitude occurs due to depolarization of the cell membrane, a change in amplitude was not observed in cells previously depolarized with K+ saline. The enhancement of Maxi-K channel activity in the presence of UTP was not diminished by Ca2+ entry blockers or by removal of extracellular Ca2+. However, in the latter case, repetitive responses progressively declined. These observations, as well as data comparing the action of low concentrations of Ca2+ ionophores (< 5 microM) to that of UTP indicate that both agents elevate cytosolic Ca2+ by mobilization of this ion from intracellular pools. However, the Ca2+ ionophore did not cause membrane depolarization, and thus did not change unitary current amplitude. The effect of UTP on Maxi-K channel activity and current amplitude was blocked by pertussis toxin and by phorbol 12-myristate 13-acetate (PMA), but was not modified by okadaic acid, or by inhibitors of protein kinase C (PKC). Our data support a model in which a pyrimidinergic receptor is coupled to a G protein, and this interaction mediates release of Ca2+ from intracellular pools, presumably via the phosphatidyl inositol pathway. This also results in activation of membrane channels that give rise to an inward current and depolarization. Ultimately, smooth muscle contraction ensues.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of phenylpropanolamine on 24-hour blood pressure in normotensive subjects administered indomethacin.

We evaluated the effect of phenylpropanolamine hydrochloride (PPA) in 14 young, healthy, normotensive women who concurrently received indomethacin. Subjects received sustained-release (SR) indomethacin 75 mg bid and were randomly assigned to receive double-blind SR PPA 75 mg/d or placebo for four days. After a six-day washout period, subjects were crossed over to the opposite four-day double-blind treatment. Following an additional six-day washout period, subjects received indomethacin placebo and PPA placebo during a final, single-blind four-day period. Twenty-four-hour blood pressure (BP) monitoring every 30 minutes and a 24-hour urine collection for prostaglandin E2 (PGE2) were performed on the fourth day of each treatment period. Compliance with the medication regimen was confirmed by drug concentrations, pill counts, and urinary PGE2 concentrations. Compared with the indomethacin and placebo treatment periods, the combination of indomethacin and PPA had no significant effect on mean systolic or diastolic BP during the 24-hour study period or during any four-hour interval. We conclude that the combination of SR PPA 75 mg/d and SR indomethacin 150 mg/d for four days has no adverse effect on BP in normotensive women.

Ca2(+)-activated K+ channels in bovine aortic smooth muscle and GH3 cells: properties and regulation by guanine nucleotides.

Research directed towards discovering agents that enhance PKCa channel activity has followed two courses. One has been defining properties of the channel, as well as making use of toxins that are specific probes for characterizing channel binding sites and for isolating and purifying the channel. The second has been a study of the regulation of PKCa channels by guanine nucleotides. The following list summarizes the major findings and conclusions that have been drawn to date. (1) The high conductance (266 +/- 11.5 pS) PKCa channel of bovine aortic smooth muscle, which is the most abundant of the 3 kinds of PKCa channels observed, has properties very similar to high conductance PKCa channels found in other cell types. (2) Charybdotoxin (ChTX), a rather specific probe for PKCa channels, has been purified to homogeneity, characterized in terms of primary sequence, labeled with 125I, and used to study binding of ChTX to the receptor/channel complex. (3) A second high affinity toxin probe, with distinct biological activity, IbTX-I, has been purified to homogeneity, and its amino acid sequence determined. One of its distinct actions is to enhance channel activity at low nM levels, and to block the channel at concentrations above approximately 5 nM. (4) A number of vasodilators having the common action of increasing cytosolic cGMP levels, have been found to enhance PKCa channel activity in on-cell patches. (5) While all of the guanine nucleotides have been noted to enhance channel activity in excised patches at high concentrations (500 uM), only GMP is effective in the 10-100 uM range. (6) These data have led us to conclude that GMP is a physiological modulator of PKCa channels, and that it is most likely the second messenger for mediating the vasodilator-induced potentiation of channel activity.

Guanosine 5'-monophosphate modulates gating of high-conductance Ca2+-activated K+ channels in vascular smooth muscle cells.

Ca2+-activated K+ channels (PKCa channels) account for the predominant K+ permeability of many types of smooth muscle cells. When activated, they oppose depolarization due to Na+ and Ca2+ channel activity. Several vasodilatory agents that increase intracellular cGMP levels (e.g., nitroprusside, adenosine, and atrial natriuretic factor) enhance the activity of these high-conductance PKCa channels in on-cell patches of bovine aortic smooth muscle cells. In addition, dibutyryl-cGMP (1.0 mM) causes a similar increase in channel activity. To pursue the mechanism of channel modulation by these agents, a series of guanine and adenine nucleotides were evaluated by using inside-out excised patches. Whereas cAMP, AMP, ADP, and ATP were ineffective, all of the corresponding guanine nucleotides potentiated PKCa channel activity when tested at a high concentration (500 microM). However, only GMP consistently enhanced channel activity in the 1-100 microM range by increasing the percent open time and frequency of opening of these channels over a wide range of potentials and Ca2+ levels without affecting single-channel conductance. Thus, GMP is a potent modulator of PKCa channels and it, rather than cGMP, may mediate the action of the vasodilators examined in this study.

Purification, sequence, and model structure of charybdotoxin, a potent selective inhibitor of calcium-activated potassium channels.

Charybdotoxin (ChTX), a protein present in the venom of the scorpion Leiurus quinquestriatus var. hebraeus, has been purified to homogeneity by a combination of ion-exchange and reversed-phase chromatography. Polyacrylamide gel electrophoresis, amino acid analysis, and complete amino acid sequence determination of the pure protein reveal that it consists of a single polypeptide chain of 4.3 kDa. Purified ChTX is a potent and selective inhibitor of the approximately 220-pS Ca2+-activated K+ channel present in GH3 anterior pituitary cells and primary bovine aortic smooth muscle cells. The toxin reversibly blocks channel activity by interacting at the external pore of the channel protein with an apparent Kd of 2.1 nM. The primary structure of ChTX is similar to a number of neurotoxins of diverse origin, which suggests that ChTX is a member of a superfamily of proteins that modify ion-channel activities. On the basis of this similarity, the three-dimensional structure of ChTX has been modeled from the known crystal structure of alpha-bungarotoxin. These studies indicate that ChTX is useful as a probe of Ca2+-activated K+-channel function and suggest that the proposed tertiary structure of ChTX may provide insight into the mechanism of channel block.

Currents carried by sodium ions through transient calcium channels in clonal GH3 pituitary cells.

The transient or T-type Ca channel currents of voltage-clamped clonal (GH3) pituitary cells were studied after blockade of Na and K channels. Removal of Ca ions from the bathing medium abolished the Ca currents and allowed Na ions to convey current through the transient channels. These Na currents (I Na,t) were activated at threshold potentials of ca. -85 mV, reached peak amplitudes (up to 2,000 pA) at -60 mV, and were 50% inactivated at holding membrane potentials of -110 to -120 mV. The time constant for inactivation of the maximal I Na,t was 12 +/- 2.4 msec (compared with 22.1 =/- 0.85 msec for maximal I Ca,t). The I Na,t was insensitive to tetrodotoxin (1 micron) and to nimodipine (200 nM) but was abolished by 100 micron external [Ca2+]. It is suggested that high affinity Ca binding sites in the channel modulate the ion selectivity and the current flow through T-type Ca channels.

Two calcium currents in a smooth muscle cell line.

Membrane currents in small cells of a smooth muscle cell line (A10) derived from embryonic rat thoracic aorta were monitored by the patch electrode whole-cell voltage clamp technique. Three currents, two divalent cation currents, and a Ca2+-activated K+ current have been observed. The latter is readily abolished pharmacologically, allowing the characterization of the divalent cation currents. With a holding potential of -50 mV, a single divalent current, which inactivates slowly, is elicited on depolarization of the membrane potential to values positive to ca. -10 mV. The second divalent cation current is only observed when the holding potential is negative to -55 mV and the membrane is pulsed to values positive to ca. -35 mV. This current is rapidly inactivating, peaking in approximately 5 ms and decaying with a t1/2 of ca. 15 ms at 0 mV when conveyed by Ba2+. The rapidly inactivating divalent cation current is depressed by substitution of Ba2+ for Ca2+ in the bathing solution and is highly insensitive to organic Ca2+ channel blockers. The slowly inactivating channel has more typical characteristics of Ca2+ channels; it is more permeable to Ba2+ than to Ca2+ and is sensitive to modulation by dihydropyridines. These data demonstrate the presence of two distinctly different Ca2+ channels in A10 cells.

Calcium currents in GH3 cultured pituitary cells under whole-cell voltage-clamp: inhibition by voltage-dependent potassium currents.

To isolate inward Ca2+ currents in GH3 rat pituitary cells, an inward Na+ current as well as two outward K+ currents, a transient voltage-dependent current (IKV) and a slowly rising Ca2+-activated current (IKCa), must be suppressed. Blockage of these outward currents, usually achieved by replacement of intracellular K+ with Cs+, reveals sustained inward currents. Selective blockage of either K+ current can be accomplished in the presence of intracellular K+ by use of quaternary ammonium ions. When IKCa and Na+ currents are blocked, the net current elicited by stepping the membrane potential (Vm) from -60 to 0 mV is inward first, becomes outward and peaks in 10-30 msec, and finally becomes inward again. Under this condition, in which both IKV and Ca2+ currents should be present throughout the duration of the voltage step, the Ca2+ current was not detected at the time of peak outward current. That is, plots of peak outward current vs. Vm are monotonic and are not modified by nisoldipine or low external Ca2+ as would be expected if Ca2+ currents were present. However, similar plots at times other than at peak current are not monotonic and are altered by nisoldipine or low Ca2+ (i.e., inward currents decrease and plots become monotonic). When K+ channels are first inactivated by holding Vm at -30 mV, a sustained Ca2+ current is always observed upon stepping Vm to 0 mV. Furthermore, substitution of Ba2+ for Ca2+ causes blockage of IKV and inhibition of this current results in inward Ba2+ currents with square wave kinetics. These data indicate that the Ca2+ current is completely inhibited at peak outward IKV and that Ca2+ conductance is progressively disinhibited as the transient K+ current declines due to channel inactivation. This suggests that in GH3 cells Ca2+ channels are regulated by IKV.

Excitation-contraction coupling: role of K-activation within the transverse tubular system.

Long-duration Ca action potentials induced in crustacean muscle fibers after prolonged exposure to quaternary ammonium ions are accompanied by attenuated tensions with unique time courses. The tensions have three phases. The initial phase, correlated with the upstroke of the spike, is a rapid increase in tension followed by relaxation to or near to resting level (on-tension). In the second phase, tension rises slowly as the spike plateau declines. The final phase is another rapid increase and decay in tension that is correlated with termination of the action potential (off-tension). To observe these tensions, fibers must be exposed to 50-100 mM tetrabutylammonium ion for about 1 hr or to lower concentrations for longer periods (e.g., 5 mM for 20-30 hr). To obtain a similar response in fibers treated with tetraethylammonium ion, higher concentrations or longer soaking periods, or both, are required. Because neither caffeine-induced tensions in intact fibers nor contractile protein and sarcoplasmic reticulum function in skinned fibers were modified by quaternary ammonium ions, their site of action appears to be limited to surface or transverse tubular system membranes, or both. The unique tensions can be explained by considering the mode by which quaternary ammonium ions block K channels in conjunction with a scheme in which activation of K channels within the transverse tubular system controls the driving force for influx of Ca ions.

Regulation of excitation-secretion coupling by thyrotropin-releasing hormone (TRH): evidence for TRH receptor-ion channel coupling in cultured pituitary cells.

The electrophysiological and secretory properties of a well-studied clonal line of rat anterior pituitary cells (GH3) have been compared with a new line of morphologically distinct cells derived from it (XG-10). The properties of the latter cells differ from the parent cells in that they do not have receptors for thyrotropin-releasing hormone and their basal rate of secretion is substantially higher (ca. three- to fivefold). While both cell types generate Ca++ spikes, the duration of the spike in XG-10 cells (ca. 500 msec) is about 2 orders of magnitude longer than that in GH3 cells (5-10 msec). The current-voltage characteristics of the two cell types are markedly different; the conductance of GH3 cells is at least 20-fold higher than XG-10 cells when cells are depolarized to more positive potentials than the threshold for Ca++ spikes (approximately -35 mV). While treatment of GH3 cells with the secretagogues tetraethylammonium chloride or thyrotropin-releasing hormone decreases the conductance in this voltage region to approximately the same as that for XG-10 cells, the electrophysiological and secretory properties of XG-10 cells are unaffected by treatment with either of these agents. Results of this comparative study suggest that XG-10 cells lack tetraethylammonium-sensitive K+ channels. The parallel loss of thyrotropin-releasing hormone receptor binding activity and of a K+ channel in XG-10 cells implies that the thyrotropin-releasing hormone receptor may be coupled with, or be an integral part of, this channel. Apparently thyrotropin-releasing hormone, like tetraethylammonium chloride, acts by inhibiting K+ channels resulting in a prolongation of the action potential, promoting Ca++ influx and subsequently enhancing hormone secretion.

A learning disability screening program in a public school.

The outline of screening program for learning disabilities in a public school is presented in this paper. The screening program consisted of four standardized measures (Information Subtest of the WPPSI, SEARCH, Evanston Early Identification Scale, Bender-Gestalt) and six of the clinical observations suggested by Ayres. Forty-three kindergarten and first graders (25 males, 18 females) were tested and each child was categorized as low, moderate, or high risk for learning disabilities on each of the measures. Approximately 10 percent of the children were identified as at high risk for learning disabilities, but the children did not perform at the same risk level on all measures. A factor analysis of the four measures and six observations revealed that three factors prevailed in this screening program; cognitive abilities, fine motor control, and reflex integration. This analysis also indicated the importance of assessing neurophysiological immaturity in a screening program. The implications of the categories used are discussed.

Fieldwork performance, academic grades, and pre-selection criteria of occupational therapy students.

To assess the relationship between pre-selection criteria, academic grades, and fieldwork performance, 45 basic master's occupational therapy students who graduated from New York University between October 1974 and October 1977 were selected. The students' undergraduate and occupational therapy records were reviewed for undergraduate grade point averages (GPA), prerequisites, major, occupational therapy grades, and Field Work Performance Report scores (FWPR). The results indicated that undergraduate GPA and occupational therapy GPA correlated with both psychosocial dysfunction and physical disability FWPR scores. However, a student's participation in a Fieldwork I experience was unrelated to the score received on the FWPR for a Fieldwork II experience. Discussion also focused on the limited usefulness of the FWPR as an evaluative tool since the mean scores were very high and its variability was rather low.

Functional heterogeneity of the sarcoplasmic reticulum within sarcomeres of skinned muscle fibers.

Precipitation of Ca oxalate in the sarcoplasmic reticulum of chemically skinned rabbit psoas fibers caused an increase in light scattering which was proportional to the amount of Ca accumulated per unit fiber volume. The increase in scattering was used to measure net accumulation rates and steady-state Ca capacities of the sarcoplasmic reticulum in single fibers. The data obtained were qualitatively and quantitatively similar to those reported for isolated vesicle preparations. Under conditions in which Ca was not depleted from the medium, Ca accumulation was linear with time over much of its course. Steady-state capacities were independent of the Ca concentration; uptake rates were half-maximal at 0.5 microM Ca++ and saturated above about 1.0 microM. Both rate and capacity varied with the oxalate concentration, being maximal at oxalate concentrations greater than or equal to mM and decreasing in proportion to one another at lower concentrations, with a threshold near 0.25 mM. At the lower loads, electron micrographs showed many sarcoplasmic reticulum elements empty of precipitate alongside others that were full, whereas virtually all were filled in maximally loaded fibers. These data indicate that the Ca oxalate capacity of each fiber varies with the number and volume of elements in which Ca oxalate crystals can form at a given oxalate concentration, and that individual regions of the sarcoplasmic reticulum within each sarcomere differ in their ability to support Ca oxalate precipitation. Our working hypothesis is that this range in ability to form Ca oxalate crystals involves differences in ability to accumulate and retain ionized Ca inside the sarcoplasmic reticulum.

Filament interaction in intact muscle fibers monitored by light scattering.

Measurement of changes in optical properties of intact muscle fibers during contraction has proven to be difficult or, in some cases, impossible due to movement of the muscle relative to the incident beam. In this paper we describe a technique for immobilizing single fibers in clear gelatin, which permits measurement of light scattering signals undistorted by movement artifacts. We also describe the phase and amplitude relationship between changes in intensity of light scattering (at 90 degrees to incident beam) and tensions induced by electrically activating single fibers. With tensions that range up to 50% Po (Po = maximal tension measured by exposure of fibers to 200 mM K+), the maximal increase in light scattering is about 25% of that for resting fibers. The scattering increase precedes tension, and at low temperatures the interval between the two peaks can be 50--100 msec. We interpret these data on intact fibers, as we did our earlier data from studied on skinned fibers, as indicating that increases in light scattering power of muscle are primarily due to attachment of myosin cross-bridges to actin filaments.

Filament interaction monitored by light scattering in skinned fibers.

The intensity of light scattered by chemically skinned rabbit psoas fibers in relaxed, rigor, and activated states was monitored at 90 degrees to the incident beam. In the relaxed state, scattering varied in proportion to the volume of muscle in the beam. Scattering increased to 2.3 times the resting value when rigor was induced by withdrawal of MgATP or when the myofibrils were activated by the caffeine-induced release of Ca from the sarcoplasmic reticulum. The rigor-induced increase in scattering decreased monotonically when MgATP was reintroduced stepwise (0-100 microM). This decrease in scattering was accompanied by an increase in tension up to an optimum MgATP level of approximately 10 microM, and then tension decreased at higher concentrations (10-100 microM). The increase in scattering during both rigor and activation was dependent upon fiber length. At lengths when thick-thin filament overlap was near zero, the light signal due to rigor and activation fell to within 10% of the signal for the relaxed fiber at that length. The signal during rigor increased only minimally (approximately 10%) when stretch (approximately 1%) was applied. This increase in signal was small despite a measured 5- to 10-fold increase in tension and an estimated twofold increase in stiffness. Thus, the increased light scattering caused by rigor and activation depends on filament overlap and not tension, stiffness, or substrate binding.

Altered calcium conductance in pawns, behavioural mutants of Paramecium aurelia.

Pawns are behavioural mutants (in one of three genes) of Paramecium aurelia that have lost, to varying degrees, the reversal response which is thought to depend on the calcium influx during excitation. This report shows that all of the single and double mutants have reduced active inward (calcium) current, the reduction correlating with the degree of behavioural deficit. All of the mutants display normal resting potential, input impedance and delayed rectification. Mutants in genes pwA and pwC show normal anomalous rectification, but pwB mutants do not show anomalous rectification until the membrane is hyperpolarized further. We suggest that the pwA gene plays a role in depolarization sensitivity (the 'gate') and the pwB gene a role affecting either the wall of the channel itself or the total number of channels.

Effects of caffeine on crayfish muscle fibers. II. Refractoriness and factors influencing recovery (repriming) of contractile responses.

When caffeine evokes a contraction, and only then, crayfish muscle fibers become refractory to a second challenge with caffeine for up to 20 min in the standard saline (5 mM K(o)). However, the fibers still respond with contraction to an increase in K(o), though with diminished tension. Addition of Mn slows recovery, but the latter is greatly accelerated during exposure of the fiber to high K(o), or after a brief challenge with high K(o). Neither the depolarization induced by the K, nor the repolarization after its removal accounts for the acceleration, which occurs only if the challenge with K had itself activated the contractile system; acceleration is blocked when contractile responses to K are blocked by reducing the Ca in the bath or by adding Mn. Recovery is accelerated by redistribution of intracellular Cl and by trains of intracellularly applied depolarizing pulses, but not by hyperpolarization. The findings indicate that two sources of Ca can be mobilized to activate the contractile system. Caffeine mobilizes principally the Ca store of the SR. Depolarizations that are induced by high K(o), by transient efflux of Cl, or by intracellularly applied currents mobilize another source of Ca which is strongly dependent upon the entry of Ca from the bathing medium. The sequestering mechanism of the SR apparently can utilize this second source of Ca to replenish its own store so as to accelerate recovery of responsiveness to a new challenge with caffeine.