Oxytocin-induced phosphorylation of myosin light chain is mediated by extracellular calcium influx in pregnant rat myometrium.

Studies of oxytocin-induced phosphorylation of myosin light chain (MLC), resulting in myometrial contraction, suggest that extracellular Ca(2+) influx is involved in its signal transduction. To explore the possibility that intracellular Ca(2+) mobilization by oxytocin may also contribute to MLC phosphorylation, we investigated the relative contributions of these Ca(2+) sources to oxytocin signal transduction in myometrium of pregnant rat. In pregnant rat myometrium, oxytocin-induced Ca(2+) influx occurs via an L-type voltage-dependent Ca(2+) channel. Treatment with verapamil, an antagonist specific for these channels, significantly diminished MLC phosphorylation observed in response to oxytocin administration without affecting the release of Ca(2+) from intracellular Ca(2+) stores. Furthermore, oxytocin-induced MLC phosphorylation was not observed when extracellular Ca(2+) was not present. Our results clearly indicate that extracellular Ca(2+) influx, rather than release from Ca(2+) storage sites, is essential for oxytocin-induced MLC phosphorylation.     

Intracellular calcium release mediated by two muscarinic receptor subtypes

Four subtypes of muscarinic acetylcholine receptor (mAChR) were stably expressed in neuroblastoma-glioma hybrid cells (NG108-15). By combining fluorescent indicator dye (fura-2) studies with electrophysiological measurements it is shown that stimulation of mAChR I and mAChR III readily leads to release of calcium from intracellular stores and to associated conductance changes, whereas stimulation of mAChR II and mAChR IV exerts no such effect. Dose-response curves describing the amplitude or the delay of the calcium rise induced by acetylcholine suggest that the apparent affinity of mAChR III for its agonist is higher by about one order of magnitude than that of mAChR I. Ionic substitution experiments and current fluctuation analysis indicate that calcium activates a K⁺-specific conductance of ‘small’ single-channel amplitude similar to the SK type [1]. Furthermore, an outward current (M current) suppressed by activation of mAChR I and mAChR III has a single-channel amplitude corresponding to a conductance of approximately 3 pS.     

In vivo imaging of oxidative stress in the kidney of diabetic mice and its normalization by angiotensin II type 1 receptor blocker

This study was undertaken to evaluate oxidative stress in the kidney of diabetic mice by electron spin resonance (ESR) imaging technique. Oxidative stress in the kidney was evaluated as organ-specific reducing activity with the signal decay rates of carbamoyl-PROXYL probe using ESR imaging. The signal decay rates were significantly faster in corresponding image pixels of the kidneys of streptozotocin-induced diabetic mice than in those of controls. This technique further demonstrated that administration of angiotensin II type 1 receptor blocker (ARB), olmesartan (5 mg/kg), completely restored the signal decay rates in the diabetic kidneys to control values. In conclusion, this study provided for the first time the in vivo evidence for increased oxidative stress in the kidneys of diabetic mice and its normalization by ARB as evaluated by ESR imaging. This technique would be useful as a means of further elucidating the role of oxidative stress in diabetic nephropathy.     

Capacitative calcium influx in human epithelial breast cancer and non-tumorigenic cells occurs through Ca2+ entry pathways with different permeabilities to divalent cations

The operation of capacitative Ca(2+) entry (CCE) in human breast cancer (SKBR3) and non-tumorigenic (HBL100) cell lines was investigated as an alternative Ca(2+) entry route in these cells. Ca(2+) readdition after thapsigargin-induced store depletion showed activation of CCE in both cell lines. SKBR3 cells exhibited retarded store depletion and CCE decay kinetics compared to the non-tumorigenic HBL100 cells, suggesting alterations in Ca(2+) homeostasis. CCE was also highly permeable to Mn(2+) and to a lesser extent to Sr(2+), but not to Ba(2+). In HBL100 cells, CCE is contributed (30%) by a Ca(2+)/Mn(2+) permeable route insensitive to low (1 microM) Gd(3+) and a Ca(2+)/Sr(2+)/Mn(2+) permeable non-selective pathway (70%) sensitive to 1 microM Gd(3+). In SKBR3 cells, the relative contribution to CCE of both routes was opposite to that in non-tumorigenic cells.     

Stimulation of Ca efflux from fura-2-loaded platelets activated by thrombin or phorbol myristate acetate

We investigated the restoration of [Ca²⁺]_i in fura-2-loaded human platelets following discharge of internal Ca²⁺ stores in the absence of external Ca²⁺. After stimulation by thrombin [Ca²⁺]_i returned from a peak level of 0.6 μM to resting levels within 4 min. When ionomycin discharged the internal stores the recovery was slower with [Ca²⁺]_i still elevated at around 0.5 μM after 5 min. Thrombin added shortly after ionomycin could accelerate the recovery of [Ca²⁺]_i and restore resting levels within 5 min, an effect that was mimicked by phorbol-12-myristate-13-acetate (PMA). Since the continued presence of ionomycin precluded reuptake into the internal stores we conclude that thrombin and PMA stimulate Ca²⁺ efflux, perhaps via protein kinase C actions on a plasma membrane Ca²⁺ pump.     

Differential coupling of alpha7 and non-alpha7 nicotinic acetylcholine receptors to calcium-induced calcium release and voltage-operated calcium channels in PC12 cells

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation channels that can modulate various neuronal processes by altering intracellular Ca(2+) levels. Following nAChR stimulation Ca(2+) can enter cells either directly, through the intrinsic ion channel, or indirectly following voltage-operated Ca(2+) channel (VOCC) activation; Ca(2+) levels can subsequently be amplified via Ca(2+)-induced Ca(2+) release from intracellular stores. We have used subtype-selective nAChR agonists to investigate the Ca(2+) sources contributing to alpha7 and non-alpha7 nAChR-mediated increases in intracellular Ca(2+) in PC12 cells. Application of the alpha7 nAChR positive allosteric modulator PNU 120596 (10 mum), in conjunction with the alpha7 nAChR agonist, compound A [(R)-N-(1-azabicyclo[2.2.2]oct-3-yl)(5-(2-pyridyl)thiophene-2-carboxamide), 10 nm], produces a rapid increase in fluo-3 fluorescence that is prevented by the selective alpha7 nAChR antagonist alpha-bungarotoxin. The non-alpha7 nAChR agonist 5-Iodo-A-85380 produces alpha-bungarotoxin-insensitive increases in intracellular Ca(2+) (EC(50) = 11.2 mum). Using these selective agonists or KCl in conjunction with general and selective VOCC inhibitors, we demonstrate that the primary route of Ca(2+) entry following either non-alpha7 nAChR activation or KCl stimulation is via L-type VOCCs. In contrast, the alpha7 nAChR-mediated response is unaffected by VOCC blockers but is inhibited by modulators of intracellular Ca(2+) stores. These results indicate that alpha7 and non-alpha7 nAChRs are differentially coupled to Ca(2+)-induced Ca(2+) release and VOCCs, respectively.     

Reduction of blood pressure by store‐operated calcium channel blockers

The voltage‐operated Ca²⁺ channels (VOCC), which allow Ca²⁺ influx from the extracellular space, are inhibited by anti‐hypertensive agents such as verapamil and nifedipine. The Ca²⁺ entering from outside into the cell triggers Ca²⁺ release from the sarcoplasmic reticulum (SR) stores. To refill the depleted Ca²⁺ stores in the SR, another type of Ca²⁺ channels in the cell membrane, known as store‐operated Ca²⁺ channels (SOCC), are activated. These SOCCs are verapamil and nifedipine resistant, but are SKF 96465 (SK) and gadolinium (Gd³⁺) sensitive. Both SK and Gd³⁺ have been shown to reduce [Ca²⁺]_i in the smooth muscle, but their effects on blood pressure have not been reported. Our results demonstrated that both SK and Gd³⁺ produced a dose‐dependent reduction in blood pressure in rat. The combination of SK and verapamil produced an additive action in lowering the blood pressure. Furthermore, SK, but not Gd³⁺ suppressed proliferation of vascular smooth muscle cells in the absence or presence of lysophosphatidic acid (LPA). SK decreased the elevation of [Ca²⁺]_i induced by LPA, endothelin‐1 (ET‐1) and angiotensin II (Ang II), but did not affect the norepinephrine (NE)‐evoked increase in [Ca²⁺]_i. On the other hand, Gd³⁺ inhibited the LPA and Ang II induced change in [Ca²⁺]_i, but had no effect on the ET‐1 and NE induced increase in [Ca²⁺]_i. The combination of verapamil and SK abolished the LPA‐ or adenosine‐5′‐triphosphate (ATP)‐induced [Ca²⁺]_i augmentation. These results suggest that SOCC inhibitors, like VOCC blocker, may serve as promising drugs for the treatment of hypertension.     

Angiotensin II and III upregulate body fluid volume of the clam worm Perinereis sp. via angiotensin II receptors in different manners

Angiotensin III (Ang III) as well as angiotensin II (Ang II) suppressed body weight loss of the clam worm Perinereis sp. under a hyper-osmotic condition, and enhanced body weight gain under a hypo-osmotic condition. Under a drying condition where the water inflow from outside the body was eliminated, Ang II suppressed body weight loss, but Ang III did not. Under these conditions, angiotensins I, IV, and (1–7) had no effect, and saralasin blocked the effects of Ang II and Ang III. It is concluded that Ang II and Ang III upregulate body fluid volume of the clam worm via Ang II receptors in different ways.     

Effects of angiotensin II antagonists on the contractile and hydrosmotic effect of AT II and AT III in the toad (Bufo arenarum)

The effects of peptide and non-peptide angiotensin II receptor antagonists on the responses to angiotensin II were examined using aortic rings and skin isolated from the toad. The contractile responses of aortic rings to (Ala-Pro-Gly) angiotensin II were inhibited by the angiotensin II analogue Leu⁸ angiotensin II, with a pA₂ value of 7.6. Similarly, the concentration response curve for (Ala-Pro-Gly) angiotensin II was displaced to the right by the specific angiotensin receptor subtype antagonist DuP 753, with a pA₂ value of 6.0. In contrast, the angiotensin receptor subtype 2 antagonists PD 123177 and CGP 42112A did not modify the contractile response to (Ala-Pro-Gly) angiotensin II. None of the antagonists was able to alter the contractile response to norepinephrine. Both Leu⁸ angiotensin II (10^-8 mol·l^-1) and DuP 753 (10^-6 mol·l^-1) partially inhibited angiotensin III-induced contractions in toad aorta. Angiotensin III, in turn, exhibited lower activity than [Asn¹-Val⁵] angiotensin II in this preparation, its molar potency ratio being 0.293. Previous work from this laboratory reported that osmotic water permeability in the skin of the toad Bufo arenarum was increased by angiotensin II, the effect being blocked by the peptide antagonist Leu⁸ angiotensin II. The hydrosmotic response to [Asn¹-Val⁵] angiotensin II (10^-7 mol·l^-1) was significantly inhibited by DuP 753 (10^-6 and 5×10^-6 mol·l^-1), whereas the response was not inhibited by a tenfold higher concentration of either PD 123177 or CGP 42112A. DuP 753 (10^-6 mol·l^-1) also inhibited the hydrosmotic response to angiotensin III (10^-7 mol·l^-1). These results suggest that receptors for angiotensin II present in isolated toad aorta and skin exhibit pharmacological features similar to those characterized as angiotensin subtype 1 in mammalian tissues.Abbreviations AT ₁ angiotensin receptor subtype 1 - AT ₂ angiotensin receptor subtype 2 - AT II angiotensin II - AT III angiotensin III - CDRC cumulative doseresponse curve(s) - NE norepinephrine - SCC short-circuit current     

Effects of salinity and turgor on calcium influx in Chara

Measurements were made of the influx of ⁴⁵Ca into internodal cells of Chara corallina in solutions containing high concentrations of NaCl. Increasing salinity in the range 4–100mol m^?3 NaCl resulted in a doubling of Ca²⁺ influx at the plasmalemma. A time-course of Ca²⁺ influx in 50 mol m^?3 NaCl, 0.5mol m^?3 CaCl₂ showed that while influx at the plasmalemma increased only 1.5-fold, influx to the vacuole increased by up to 15-fold. This was interpreted as being due to inhibition of active Ca²⁺ efflux from the cell. The stimulation of Ca²⁺ influx by increasing salinity appeared to be principally a response to reduced turgor since similar stimulations were obtained when turgor was reduced by NaCl, Na₂SO₄ or mannitol. When cells were plasmolysed Ca²⁺ influx increased by 10–20-fold. The increased permeability was relatively specific for Ca²⁺ and was inhibitable by La³⁺. Survival of cells in high salt conditions was increased by 30 mmol m^?3 La³⁺, which inhibited Ca²⁺ influx. Paradoxically, survival can also be extended by increasing external Ca²⁺ which leads to a higher influx. Therefore, it seems unlikely that the ameliorative effect of Ca²⁺ on the sensitivity of plants to high NaCl is mediated by Ca²⁺ entry across the plasmalemma. It seems more likely that the principal role of Ca²⁺ under these conditions is exerted externally through the control of membrane voltage and permeability.     

CB1 receptors diminish both Ca(2+) influx and glutamate release through two different mechanisms active in distinct populations of cerebrocortical nerve terminals

We have investigated the mechanisms by which activation of cannabinoid receptors reduces glutamate release from cerebrocortical nerve terminals. Glutamate release evoked by depolarization of nerve terminals with high KCl (30 mmol/L) involves N and P/Q type Ca(2+)channel activation. However, this release of glutamate is independent of Na(+) or K(+) channel activation as it was unaffected by blockers of these channels (tetrodotoxin -TTX- or tetraethylammonium TEA). Under these conditions in which only Ca(2+) channels contribute to pre-synaptic activity, the activation of cannabinoid receptors with WIN55,212-2 moderately reduced glutamate release (26.4 +/- 1.2%) by a mechanism that in this in vitro model is resistant to TTX and consistent with the inhibition of Ca(2+) channels. However, when nerve terminals are stimulated with low KCl concentrations (5-10 mmol/L) glutamate release is affected by both Ca(2+) antagonists and also by TTX and TEA, indicating the participation of Na(+) and K(+) channel firing in addition to Ca(2+) channel activation. Interestingly, stimulation of nerve terminals with low KCl concentrations uncovered a mechanism that further inhibited glutamate release (81.78 +/- 4.9%) and that was fully reversed by TEA. This additional mechanism is TTX-sensitive and consistent with the activation of K(+) channels. Furthermore, Ca(2+) imaging of single boutons demonstrated that the two pre-synaptic mechanisms by which cannabinoid receptors reduce glutamate release operate in distinct populations of nerve terminals.     

Spike‐dependent calcium influx in dendrites of the cricket giant interneuron

Identified wind‐sensitive giant interneurons in the cricket's cercal sensory system integrate cercal afferent signals and release an avoidance behavior. A calcium‐imaging technique was applied to the giant interneurons to examine the presence of the voltage‐dependent Ca²⁺ channels (VDCCs) in their dendrites. We found that presynaptic stimuli to the cercal sensory nerve cords elevated the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in the dendrites of the giant interneurons. The dendritic Ca²⁺ rise coincided with the spike burst of the giant interneurons, and the rate of Ca²⁺ rise depended on the frequency of the action potentials. These results suggest that the action potentials directly caused [Ca²⁺]_i increase. Observation of the [Ca²⁺]_i elevation induced by depolarizing current injection demonstrates the presence of the VDCCs in the dendrites. Although hyperpolarizing current injection into the giant interneuron suppressed action potential generation, EPSPs could induce no [Ca²⁺]_i increase. This result means that ligand‐gated channels do not contribute to the synaptically stimulated Ca²⁺ elevation. On the other hand, antidromically stimulated spikes also increased [Ca²⁺]_i in all cellular regions including the dendrites. And bath application of a mixture of Ni²⁺, Co²⁺, and Cd²⁺ or tetrodotoxin inhibited the [Ca²⁺]_i elevation induced by the antidromic stimulation. From these findings, we suppose that the axonal spikes antidromically propagate and induce the Ca²⁺ influx via VDCCs in the dendrites. The spike‐dependent Ca²⁺ elevation may regulate the sensory signals processing via second‐messenger cascades in the giant interneurons. © 2000 John Wiley & Sons, Inc. J Neurobiol 44: 45–56, 2000     

Intronic enhancement of angiotensin II type 2 receptor transgene expression in vitro and in vivo

The angiotensin II type 2 receptor (AT2R) can influence a variety of intracellular signaling molecules and cellular functions. However, its physiological functions and the roles of introns in the regulation of its expression have not been well determined. We first demonstrated that both intron 1 and intron 2 of AT2R could significantly enhance AT2R overexpression. Thus, we have provided some new prerequisites for further studies on the mechanisms that control AT2R gene expression. Next, we established a highly efficient method of delivering this receptor in vitro and in vivo using an AT2R recombinant adenoviral vector containing two introns of the AT2R. The vector may be useful in helping to uncover AT2R physiological functions and also for gene therapy related to AT2R. Moreover, we determined the important role of introns in gene expression cassettes and the inconsistency of expression between the targeted gene and the reporter gene.     

Free calcium ion concentration in the sieve-tube sap of Ricinus communis L. seedlings

Changes in free Ca²⁺ in sieve-tube sap have been proposed to be important in the regulation of phloem transport, and Ca²⁺-activated protein kinase activity has been described in phloem exudate (S.A. Avdiushko et al. 1997 J Plant Physiol 150: 552–559). Using atomic absorption spectrometry, we have determined that the total Ca²⁺ concentration in sieve-tube sap from Ricinus seedlings containing the endosperm is about 100 μM (range 80–150 μM). We used three independent methods to determine the free calcium ion concentration in the phloem sap ([Ca²⁺]_p). The first method was to calculate [Ca²⁺]_p from the total Ca²⁺ concentration, in combination with the binding constants and concentrations of the ionic solutes in phloem sap. The resultant estimate of [Ca²⁺]_p was 63 μM. The second method used the Ca-specific fluorescent dye 2-[2-(5-carboxy)oxazole]-5-hydroxy-6-aminobenzofuran-N,N,O-triacetic-acid (FURAPTRA) on exuded sieve-tube sap. Although the sap interfered severely with the fluorescence properties of the dye, Ca²⁺ titrations enabled a value of [Ca²⁺]_p = 20 μM to be deduced. The third method used Ca²⁺-selective microelectrodes on exuded sap samples, which gave an average value for [Ca²⁺]_p = 13 μM. No significant change in this value was observed during the sap exudation period. The Ca²⁺ buffer capacity was determined and the result of about 0.6 mmol · l⁻¹ · pCa⁻¹ displayed excellent agreement with the measured values of free and total Ca²⁺ concentration in sieve-tube sap. Since the measured values for free Ca²⁺ are 20- to 100-fold higher than those usually reported for the cytosol of a range of plant cells in resting conditions, it is concluded that either regulation of [Ca²⁺]_p is of limited physiological importance, or that the Ca²⁺-dependent proteins respond only to relatively high [Ca²⁺]_p. The implications for regulation of cytosolic free Ca²⁺ in symplastically connected companion cells is discussed. Received: 15 February 1998 / Accepted: 14 March 1998     

Angiotensin II inhibits the accumulation of cyclic AMP produced by glucagon but not its metabolic effects

Studies in model systems, as well as observations in intact cells, suggest that osmotic swelling of secretory granules is an essential step in exocytosis. A model is proposed whereby the low pH recorded in most secretory organelles could provide the driving force for granule swelling. The model assumes that during stimulation an exchange of H+ for alkali cations is triggered across the granule membrane. The outgoing H+ are rapidly replaced by the internal buffering capacity with a concomitant osmotic gain. The exchange is independent of anions and could be triggered by cytoplasmic Ca2+. The H+ -pump is responsible for the delta pH but independent of the exchange mechanism.     

Determination of the structure of lipid vesicle-bound angiotensin II and angiotensin I

A mass spectrometry (MS)-based strategy was developed to determine the structure of lipid vesicle-bound angiotensin II (AII) and angiotensin I (AI). It involves hydrogen-deuterium exchange (HDX), chemical modifications (e.g., nitration of tyrosine, acetylation of free amino group), and ladder sequencing. HDX is also combined with tandem mass spectrometry (MS/MS) to provide structural details at individual amino acid residues. It was observed that a major portion of both of these peptide hormones interacts with the phospholipid head groups on the surface of the vesicles and that Tyr residue is embedded in the vesicles. Both peptides have a U-shaped structure in the lipid environment.     

Effects of phorbol esters and a calcium ionophore on angiotensin II binding in rat brain synaptosomes

In previous studies we determined that protein kinase C (PKC) and calcium are important intracellular regulators of neuronal angiotensin II (Ang II) binding sites. In the present study we investigated the effects of the protein kinase C (PKC) agonist phorbol esters (PE) and also a calcium ionophore (A23187) on the specific binding of [¹²⁵I]Ang II to brain synaptosomes prepared from rats of different ages. The rationale was to determine whether the larae changes in the level of brain Ang II specific binding observed in different age rats are due to changes in the regulation of these sites by PKC or by calcium. The present data indicate no qualitative differences in the effects of PE or A23187 on [¹²⁵I]Ang II specific binding to hypothalamic or brain stem synaptosomes, from either 2–5 or 70-day-old rats, i.e. the active PE TPA increased while A23187 decreased Ang II binding in all situations. Thus, the dramatic differences in brain Ang II specific binding seen with age appear not to be due to changes in regulation by PKC or calcium.     

Role of Ras in ethanol modulation of angiotensin II activated p42/p44 MAP kinase in rat hepatocytes

Angiotensin II plays a role in both liver cell proliferation and liver injury but the effects of ethanol on angiotensin II signaling in liver are not clearly understood. We have investigated the role of Ras in ethanol modulation of p42/p44 mitogen-activated protein kinase (MAPK) stimulated by angiotensin II (Ang II) in primary cultures of rat hepatocytes. Hepatocytes were incubated with ethanol (100 mM) for 24 h, then stimulated with Ang II (100 nM). The level of p42/p44 MAPK phosphorylation was measured by Western blot analysis and Ras activation was assessed by specific binding of Ras-GTP (activated form) to a GST-RBD fusion protein containing Ras-binding domain (RBD) of Raf-1. Ethanol potentiated p42/p44 MAPK activation by Ang II, whereas ethanol alone did not significantly affect phosphorylation of p42/p44 MAPK. Ang II increased Ras activity by about 2 fold. Ethanol exposure increased Ang II stimulated Ras activity by an additional about 2 fold. Ethanol alone elicited a small increase in basal Ras activity. Pretreatment with manumycin A (10 microM), a Ras farnesylation inhibitor, partially blocked both Ang II-activated and ethanol-potentiated MAPK activities. These data provided the first evidence that ethanol potentiation of Ang II stimulated p42/p44 MAPK is mediated, in part, by Ras in hepatocytes.