45Ca Movements Research Articles

Effects of chlorbutol on 45Ca movements and contractile responses of rat aorta and its relevance to the actions of Syntocinon.

The effects of chlorbutol (0.7, 1.4 and 2.8 mM) on the contractile responses induced by KCl and noradrenaline (NA) and on 45Ca movements have been studied on rat isolated thoracic aorta. Chlorbutol decreased, in a dose-dependent manner, contractions induced by KCl and NA and this effect was observed whether it was added before or after the induced contractions. Preincubation with chlorbutol inhibited the contractile responses elicited by addition of Ca (1-5 mM) to Ca-free high-potassium solution. It also inhibited in a dose-dependent manner the 45Ca influx but increased 45Ca efflux in rat aortic strips. These results suggest that chlorbutol decreases peripheral resistance by reducing the availability of intracellular Ca to the contractile machinery in vascular smooth muscle cells. The effects of synthetic oxytocin (Syntocinon) at concentrations containing the same chlorbutol concentration were quantitatively similar from those produced by chlorbutol alone. Therefore, the inhibitory cardiovascular effects ascribed previously to synthetic oxytocin may be attributed to its preservative, chlorbutol, and not to oxytocin itself.

Acute effect of prolactin on bone 45Ca accumulation in rats.

In an attempt to evaluate the acute effect of PRL on bone turnover, experiments were performed in sexually mature female Wistar rats which were given an intraperitoneal (i.p.) administration of 1.25 mM CaCl2 solution containing 6 microCi 45Ca. Dose response study showed a two fold higher rate of 45Ca uptake in 30 min by control trabecular bones (sternum and vertebrae 5-6) compared with control compact bones (femur and tibia). Femur exhibited a dose dependent increase in 45Ca uptake in response to pharmacological doses of 0.01 and 0.02 mg PRL/100 g body weight but was less responsive to 0.04 mg PRL/100 g body weight. Tibia only responded with increased 45Ca uptake to 0.02 and 0.04 mg PRL/100 g body weight while trabecular bones were unresponsive. By varying the intervals between administration of 45Ca (0 min) and PRL (0, 30 or 60 min) and bone harvesting (30, 60 or 90 min), it was found that 0.01 mg PRL/100 g body weight had a biphasic action on 45Ca movement. It initially enhanced 45Ca influx into femur by 30 min followed by an accelerated 45Ca efflux from femur back to the circulation. It could be concluded that PRL acutely stimulated calcium turnover in compact bone.

Effects of propafenone on 45Ca movements and contractile responses in vascular smooth muscle

1. In rat isolated aorta the class Ic antiarrhythmic drug, propafenone, dose-dependently inhibited the contractile responses induced by high K (80 mM) and noradrenaline (NA, 10(-5) M), the IC50s being 2.5 +/- 0.7 x 10(-6) M and 8.7 +/- 0.8 x 10(-6) M, respectively. These inhibitory actions were also observed with propafenone added after the induced contractions. 2. Contractile responses induced by addition of Ca to 0 Ca high-K solution were also inhibited by propafenone (IC50 = 2.5 +/- 0.8 x 10(-6) M). Moreover, propafenone inhibited the contractile responses elicited by NA in strips incubated in 0 Ca (IC50 = 1.9 +/- 0.9 x 10(-6) M). 3. Propafenone also inhibited (IC50 = 1.2 +/- 0.4 x 10(-5) M) the development of spontaneous mechanical activity in portal vein segments. 4. Propafenone, 5 x 10(-6) M and 10(-5) M, inhibited 45Ca uptake stimulated by high K or NA without altering 45Ca uptake in resting strips. 5. These results indicated that in rat isolated aortae and portal veins propafenone inhibited Ca entry through voltage-operated channels and NA-induced Ca uptake as well as Ca release from intracellular stores. As a consequence it would reduce the concentration of intracellular free Ca available at the contractile apparatus for vascular smooth muscle contraction.

Hyperreactivity and 45Ca movements in sensitized guinea-pig tracheal muscle

Responses to KCl and histamine and 45Ca movements were studied in trachea from normal and actively sensitized guinea-pigs. Sensitized tracheas were hyperresponsive and hypersensitive to KCl and histamine. 45Ca uptake experiments show that sensitized tracheal muscle behaves as normal except that the uptake of 45Ca in low concentration (0.03 mM) Ca2+ solution was higher and the number of binding sites for the high affinity component of 45Ca uptake (as estimated by Scatchard-coordinate plot) was augmented. Additionally, in sensitized tracheal muscle, incubation in low (0.03 mM) Ca2+ solution followed by La3+ wash-out resulted in a greater amount of residual 45Ca than in normal tissues. KCl, but not histamine, increased the La3+ resistant 45Ca content. This increase was greater in sensitized than in normal trachea. This demonstrates the existence of hyperreactivity and altered 45Ca movements in sensitized trachealis muscle.

Ca2+ absorption in the pars recta of cortical S2 rabbit proximal tubules: role of diffusion.

Individual partes rectae of cortical S2 proximal tubules were dissected from rabbit kidneys and perfused in vitro. The bath was a protein-free, plasmalike solution, whereas the perfusate simulated "late" proximal tubular fluid (high [Cl-], low [HCO3-], absence of Na+-cotransported solutes). Transepithelial voltages at the perfusion and collection ends were measured simultaneously in doubly cannulated tubules. Ionized calcium concentrations in perfusates, collectates, and the bath were calculated from measurements of calcium ion activities made in situ with Ca2+-selective microelectrodes. Epithelial calcium permeability was estimated from the bath-to-lumen movement of 45Ca. Total calcium concentrations of perfused and collected fluids were measured by continuous-flow microcolorimetry. Under these conditions there was a lumen-positive transepithelial voltage that was equal at both ends of the tubules. Luminal [Ca2+] [corrected], which decreased along the length of the tubule, was greater than that of the bath. Using the measured calcium permeability, we calculated the diffusional component of the net calcium absorptive flux from the mean transepithelial delta [Ca2+] and the measured transepithelial voltage. We found that ionic diffusion accounted for the net flux, suggesting that diffusion is the primary mechanism of calcium absorption in this segment of the rabbit nephron.

Effects of oxodipine on 45Ca movements and contractile responses in vascular smooth muscle.

1. The inhibitory effects of oxodipine, a new dihydropyridine, were compared with those of nifedipine on contractile responses in rat isolated aortic strips and on spontaneous mechanical activity in portal vein segments. 2. In rat isolated aorta oxodipine (IC50 = 7.8 +/- 1.8 x 10(-9)M) and nifedipine (IC50 = 8.5 +/- 2.5 x 10(-9)M) dose-dependently inhibited the contractile responses induced by high K (80 mM), whereas responses to noradrenaline (NA, 10(-6)M) were only slightly affected (IC50 greater than 10(-7)M). These inhibitory actions were observed with both drugs added either before or after the induced contractions. 3. Contractile responses induced by addition of Ca to 0Ca high-K solution were also dose-dependently inhibited by oxodipine (IC50 = 4.5 +/- 2.5 x 10(-9)M). However, oxodipine up to 10(-6)M did not modify the contractile responses obtained in strips incubated in 0Ca when Ca was added in the presence of NA. 4. Oxodipine and nifedipine also inhibited the development of spontaneous mechanical activity in portal vein segments. 5. Oxodipine inhibited 45Ca uptake stimulated by high K (I50 = 8.7 +/- 2.5 x 10(-9)M) or by NA (I50 greater than 10(-7)M). However, it did not modify 45Ca uptake and 45Ca efflux in resting strips or 45Ca efflux stimulated by NA. 6. These results indicate that the effects of oxodipine on vascular smooth muscle may be due to the blockade of Ca entry through potential- and receptor-operated channels; it was at least 10 times more selective for potential-operated channels. Oxodipine did not modify Ca entry through passive leak channels and NA-induced intracellular Ca release.

Superoxide anion production in glass-adherent polymorphonuclear leukocytes and its relationship to calcium movement.

The production of superoxide anion (O2-.) was measured in relation to 45Ca movement in glass-adherent polymorphonuclear leukocytes (PMNs), and the results were compared with those obtained by ourselves and others on PMNs in suspension. In adherent PMNs, O2-. production stimulated by N-formyl-methionyl-leucyl-phenylalanine (FMLP) was of rapid onset and short duration; this was also true of PMNs in suspension. However, O2-. production was insensitive to the concentration of extracellular calcium. Both in adherent and non-adherent PMNs, O2-. production stimulated with phorbol myristate acetate (PMA) had a latency time and was of long duration. In adherent PMNs, pretreatment with PMA potentiated the FMLP-induced O2-. production by lengthening its duration without changing its initial rate. In adherent PMNs (10(-10)-10(-7) M) FMLP induced a fast but transient dose-dependent increase in 45Ca within 1 min, whereas PMA only released 45Ca about 5 min after its addition to the cell culture medium. Pretreatment of PMNs with 10 or 100 ng/ml PMA for 3 min before stimulation by 10(-7) M FMLP reduced the 45Ca efflux observed with FMLP alone. We conclude that O2-. production by adherent PMNs cannot simply be related to Ca2+ movement. In comparison with non-adherent cells, adherence seemed to interfere with the characteristics of both calcium and O2-. generation, probably by modifying the cytoskeleton.

Effects of BRL 34915, a putative K channel opener, on transmembrane 45Ca movements in rabbit aortic smooth muscle

The effects of BRL 34915, a putative K channel opener, on transmembrane Ca 2+ movements were studied in isolated rabbit aortic strips, using the 45Ca flux techniques. BRL 34915 (1 and 10 μM) inhibited the contraction and Ca entry in response to norepinephrine (NE) and angiotensin II, each at 0.3 μM. BRL 34915 differed from Na nitroprusside in that it demonstrated little or no inhibition on the contraction and 45Ca efflux provoked by NE in Ca 2+-free buffer, suggesting that the effect of BRL 34915 on intracellular Ca release plays a minor role in inhibiting the vasoconstrictor responses to receptor agonists. Moreover, BRL 34915 (100 μM) did not stimulate cyclic GMP formation in rabbit aortic slices. BRL 34915 (10 μM) failed to inhibit the vascular responses to KCl (20 and 40 mM). Tetraethylammonium (20 mM), a K channel blocker, abolished the vasorelaxant action of BRL 34915 against NE. However, Na nitroprusside inhibition of NE contraction was similarly affected. It is concluded that reduction of Ca entry appears to be the predominant mechanism mediating the inhibitory effect of BRL 34915 on the contractile responses to vasoconstrictor stimuli.

Effect of milrinone on contractility and 45Ca movements in the isolated rabbit aorta

The effects of milrinone on 45Ca movements and on contractile responses were studied in rabbit aortic smooth muscle. Milrinone, 10 -7−5×10 -4 M, inhibited the responses induced by high K and noradrenaline (NA). These effects were observed when milrinone was added before or after the induced contractions and were similar in aortas with or without endothelium. The Ca-induced contractions of K-depolarized aortas as well as the contractions induced by caffeine in normal and in Ca-free solution were also inhibited dose dependently by milrinone. Milrinone reduced the 45Ca influx in resting or non-stimulated aortic rings as well as in aortic rings stimulated by NA. On the other hand, neither the 45Ca influx stimulated by high K or the 45Ca efflux stimulated by NA were altered by pretreatment with milrinone. It is concluded that milrinone inhibits vascular smooth muscle contractility by probably acting at multiple sites to decrease the availability of intracellular free Ca required for activation.

Trans-sarcolemmal Ca2+ movements associated with contraction of the rabbit right ventricular wall.

The purpose of this study was to examine the movements of Ca2+ into the myocardium from the extracellular space during the course of muscle contraction. Equilibration of the rabbit right ventricular wall with perfusate containing 45Ca was measured by collecting effluent droplets over time. This protocol was carried out in a quiescent muscle and then repeated 15-20 minutes later in an identical fashion except that halfway through the collection period the muscle was stimulated to contract. We were thus able to quantitate the contraction-dependent changes in 45Ca movements. In control experiments using [58Co]EDTA as an extracellular space marker, we observed that contraction altered the volume of this space. This alteration in extracellular space was relatively small, and the quantitation of 45Ca movements was corrected for this change. The addition of 1 microM Bay K 8644 to the perfusate stimulated tension and augmented Ca2+ depletion from the extracellular space. The addition of 15 microM nifedipine to the perfusate significantly reduced both tension development and Ca2+ depletion from the extracellular space of the muscle. Net contraction-dependent movement of Ca2+ from the extracellular space into the myocardium under control conditions at 1 mM [Ca2+] was 10-14 mumol Ca2+/kg tissue wet wt/beat. This value indicates either a large contribution of Ca2+-induced Ca2+ release from the sarcoplasmic reticulum and/or significant contribution of sarcolemmal bound Ca2+ to excitation-contraction coupling in the rabbit ventricle.

Modification by decreased temperature and hypoxia of 45Ca movements in stimulated smooth muscle of rabbit aorta

1. 1. In rabbit aortic smooth muscle, contractile responsiveness to 10 −6 M norepinephrine or 60 mM added K + was inhibited by hypoxia (induced by 100% N 2) or decreased bath temperature. 2. 2. Hypoxia increased K +-induced 45Ca efflux, inhibited K +-induced 45Ca uptake, and inhibited norepinephrine-induced 45Ca release; lowering bath temperature by 10°C decreased resting 45Ca uptake and norepinephrine-induced 45Ca release by 30–40% and decreased the 45Ca uptake elicited with norepinephrine or K + by more than two-thirds. 3. 3. Thus, hypoxia and temperature variations affect different Ca 2+ components. Hypoxia decreases the norepinephrine-sensitive membrane Ca 2+ fraction and the K +-stimulated mitochondrial Ca 2+ fraction whereas decreased temperature most strongly inhibits membrane-associated Ca 2+ uptake increases elicited with either norepinephrine or high K +.

Biological activity of vitamin D metabolites and analogs: Dose-response study of 45Ca transport in an isolated chick duodenum perfusion system

We have previously reported that vascular perfusion of the normal vitamin D 3-replete chick duodenum with physiological amounts of 1,25-dihydroxy vitamin D 3 [1,25(OH) 2D 3] increases the unidirectional movement of 45Ca from the lumen to the venous effluent under conditions of normal (0.9 mM) Ca 2+ concentrations in both the lumen and vascular perfusate [ Endocrinology 115: 1476 1984)]. The purpose of the present study was to determine the dose responsivity of this perfused intestinal calcium transport system for 1,25(OH) 2D 3 and some structurally related congeners. The dose-response curve was biphasic for all compounds studied; for 1,25(OH) 2D 3 initial stimulation of transport was detected at only 30 pM (the plasma concentration of 1,25(OH) 2D 3 is normally 125 pM] while maximal stimulation was 154% above control at a concentration of 650 pM. Above 650 pM 1,25(OH) 2D 3 the stimulation fell off sharply and transport had returned to basal levels by 1.3 nM. The relative potency of the D homologs tested was respectively 1,25(OH) 2D 3: 10,000; 1-alpha-hydroxy vitamin D 3: 400; 25-hydroxyvitamin D 3: 200; 24R,25-dihydroxy-vitamin D 3: 137; vitamin D 3: 34; 5,6- trans-25-hydroxyvitamin D 3: 3. These results establish the usefulness of the perfused intestinal calcium transport system to study the nongenomic actions of 1,25(OH) 2D 3 on intestinal calcium transport.

Calcium transport across the pars recta of cortical segment 2 proximal tubules.

Partes rectae of cortical segment 2 proximal tubules were dissected from rabbit kidneys and perfused in vitro. Ca concentrations of perfused and collected fluids were measured by continuous-flow microcolorimetry. Epithelial Ca permeability (P) was estimated from the bath-to-lumen movement of 45Ca. The transepithelial voltage (psi) and [Ca2+] difference were varied simultaneously by changing perfusate composition. Tubules that were perfused and bathed with an identical artificial ultrafiltrate of plasma displayed a lumen-negative psi, a collectate [Ca] greater than perfusate, and net Ca secretion. Tubules perfused with "late" proximal tubule fluid (high [Cl], low [HCO3], low concentrations of Na+-cotransported solutes) demonstrated a lumen-positive psi, a perfusate [Ca2+] greater than the bath, a collectate [Ca] less than perfusate, and net Ca absorption. Under each of these conditions, net Ca flux was in the direction predicted by the experimentally measured driving forces for diffusional Ca transport. Tubules that were cooled while being perfused with late proximal tubule fluid showed an increased lumen-positive psi but reduced net Ca absorption. The latter finding was consistent with reduced Ca ion diffusion related to a smaller P at the lower temperature. I conclude that Ca2+ diffusion is an important component of net Ca absorption in this segment of the nephron.

The effect of oxytocin on contractile responses and 45Ca movements in rat isolated aortic strips.

The effects of oxytocin (Oxt) on contractile responses and transmembrane Ca fluxes were studied in rat isolated aortic strips. Oxt (50-1000 mu ml-1) induced a dose-dependent contractile response and spontaneous myogenic activity. The Oxt-induced contractile response was not inhibited by pretreatment of aortic strips with phentolamine plus practolol, atropine, diphenhydramine plus cimetidine or indomethacin, but was significantly reduced by verapamil or deamino-ethyl-Oxt. The dose-response curve for Oxt was shifted upwards and to the left by increasing the Ca concentration of the medium from 1.8 to 4 mM. Oxt also shifted upwards the dose-response curve to Ca (1 to 5 mM) in aortic strips incubated in K depolarizing Ca-free media. Oxt increased the La3+-resistant 45Ca content without altering 45Ca efflux. The present results suggest that in rat isolated aortic strips Oxt produces a contractile response which, as previously found with other neurotransmitters, can be partly attributed to an increase of Ca influx through receptor-operated channels and to the release of Ca from intracellular stores.

Axonal transport and the movement of 45Ca inside the giant axon of squid

45Ca was microinjected directly into the giant axon of squid, and the radioisotope profile along the axon was determined after 2–12 h. Our results indicated that the intracellular Ca ions at the axon, unlike those at the cell body, were not axonally transported at a fast rate. The implication of this finding on the involvement of Ca in the axonal transport system is discussed.

Effects of pincainide on contractile responses and 45Ca movements in rat isolated vascular smooth muscle

The effects of pincainide, a new β-amino anilide, on contractile responses and 45Ca fluxes were studied in rat aorta and on spontaneous mechanical activity in rat portal veins. Pincainide (10 −5 to 10 −2 M) produced a dose-dependent inhibition of noradrenaline (NA) and high K +-induced contractions. These inhibitory effects were observed with pincainide added either before or after the induced contractions. The Ca 2+-induced contractions of K +-depolarized aorta as well as the spontaneous mechanical activity of portal veins were also inhibited by pincainide. Pincainide (5 × 10 −3 M) inhibited the 45Ca influx stimulated by 10 −6 M NA and increased 45Ca efflux. It was concluded that in isolated rat aorta, pincainide not only inhibited the influx of Ca 2+ reducing the contractile responses to NA and high K + but it also inhibited other effects related to NA-induced release of intracellular Ca 2+ stores.

P-79) - Measurements of 45Ca movements in enzymic dispersed single intact and skinned muscle cells of the porcine coronary artery

P-79) - Measurements of 45Ca movements in enzymic dispersed single intact and skinned muscle cells of the porcine coronary artery

The action of tabernanthine on noradrenaline-stimulated contractions and 45Ca movements in rat isolated vascular smooth muscle

The iboga alkaloid tabernanthine inhibited depolarization (100 mM K +)-induced contractions in aorta and mesenteric arteries in an concentration-dependent manner with the respective IC 50 values being about 21 and 7 μM. Contractions elicited by noradrenaline in the mesenteric artery were potentiated by lower concentrations (0.1 and 1.0 μM) of tabernanthine while they were inhibited by higher concentrations (10–100 μM). Tabernanthine produced only inhibition of noradrenaline-elicited responses in aorta and portal vein, the vein being the most sensitive of the vessels to the inhibitory effects of the compound. The magnitude of the spontaneous contractions of the portal vein, the aortic intracellular calcium fraction releasable by noradrenaline and the turnover of calcium in unstimulated aorta were enhanced by tabernanthine. Depolarization-stimulated 45Ca influx and contractions in the aorta were inhibited to a similar extent by tabernanthine and 100 μM virtually abolished the 45Ca influx stimulated by noradrenaline and depolarization. It is concluded that tabernanthine has a calcium entry blocking action but also has other actions related to the turnover of intracellular calcium releasable by noradrenaline.

Infiltration and Movement of 45Ca into Potatoes

Abstract Immediately following vacuum infiltration of 45Ca into ‘Katahdin’ potatoes (Solanum tuberosum L.) the label was distributed within the tuber in the following manner: 45%, 20%, and 35% in the stem end, bud end, and middle section, respectively. Initially, the bulk of the radioactive calcium was localized 0–5 mm below the periderm layer. However, over a 60–day period, the calcium moved toward the center of the tuber. Temperature had no effect on this process.

Is the red cell calcium pump electrogenic?

1. In inside-out vesicles of high potassium permeability, prepared from human red cell membranes, volume changes accompanying the action of the Ca2+ pump were measured by recording the intensity of light scattered by a suspension of these vesicles. Replacing Cl- by the impermeant gluconate anion changed swelling into shrinking. 2. Assuming that in Cl- media two Cl- ions accompany one Ca+ ion moved by the pump and in gluconate media two K+ ions are exchanged for one Ca2+ ion resulted in a good agreement between relative Ca2+ transport rate obtained from the volume change and from direct measurement of 45Ca uptake in the two media. 3. The fact that it is possible to change co-transport of Ca with Cl- into counter-transport of Ca2+ for K+ rules out that within the pump there is an obligatory coupling of Ca2+ movement with movement of another ion species (including the proton). The conclusion, therefore, is that the Ca2+ pump must be electrogenic. 4. The combination of measurement of volume change with direct measurement of 45Ca movement yielded 5-6 microliter/mg protein for the volume of the vesicles.