Oscillatory Cl current induced by angiotensin II in rat pulmonary arterial myocytes: Ca dependence and physiological implication

We have previously reported that angiotensin II (ANG II) induces oscillations in the cytoplasmic calcium concentration ([Ca²⁺]_i) of pulmonary vascular myocytes. The present work was undertaken to investigate the effect of ANG II in comparison with ATP and caffeine on membrane currents and to explore the relation between these membrane currents and [Ca²⁺]_i. In cells clamped at −60 mV, ANG II (10 μM) or ATP (100 μM) induced an oscillatory inward current. Caffeine (5 μM) induced only one transient inward current. In control conditions, the reversal potential (E_rev) of these currents was close to the equilibrium potential for Cl⁻ ions (E_Cl = −2.1 mV) and was shifted towards more positive values in low-Cl⁻ solutions. Niflumic acid (10–50 μM) and DIDS (0.25-1 mM) inhibited this inward current. Combined recordings of membrane current and [Ca²⁺]_i by Indo-1 microspectrofluorimetry revealed that ANG II- and ATP-induced currents occurred simultaneously with oscillations in [Ca²⁺]_i, whereas the caffeine-induced current was accompanied by only one transient increase in [Ca²⁺]_i Niflumic acid (25 μM) had no effect on agonist-induced [Ca²⁺]_i responses, whereas thapsigargin (1 μM) abolished both membrane current and the [Ca²⁺]_i response. Heparin (5 mg/ml in the pipette solution) inhibited both [Ca²⁺]_i responses and membrane currents induced by ANG II and ATP, but not by caffeine. In pulmonary arterial strips, ANG II-induced contraction was inhibited by niflumic acid (25 μM) or nifedipine (1 μM) to the same extent and the two substances did not have an additive effect. This study demonstrates that, in pulmonary vascular smooth muscle, ANG II, as well as ATP, activate an oscillatory calcium dependent chloride current which is triggered by cyclic increases in [Ca²⁺]_i and that both oscillatory phenomena are primarily IP₃ mediated. It is suggested that ANG II-induced oscillatory chloride current could depolarise the cell membrane leading to activation of voltage-operated Ca²⁺ channels. The resulting Ca²⁺ influx contributes to the component of ANG II-induced contraction that is equally sensitive to chloride or calcium channel blockade.     

Return Customers: Foraging Site Fidelity and the Effect of Environmental Variability in Wide-Ranging Antarctic Fur Seals

Strategies employed by wide-ranging foraging animals involve consideration of habitat quality and predictability and should maximise net energy gain. Fidelity to foraging sites is common in areas of high resource availability or where predictable changes in resource availability occur. However, if resource availability is heterogeneous or unpredictable, as it often is in marine environments, then habitat familiarity may also present ecological benefits to individuals. We examined the winter foraging distribution of female Antarctic fur seals, Arctocephalus gazelle, over four years to assess the degree of foraging site fidelity at two scales; within and between years. On average, between-year fidelity was strong, with most individuals utilising more than half of their annual foraging home range over multiple years. However, fidelity was a bimodal strategy among individuals, with five out of eight animals recording between-year overlap values of greater than 50%, while three animals recorded values of less than 5%. High long-term variance in sea surface temperature, a potential proxy for elevated long-term productivity and prey availability, typified areas of overlap. Within-year foraging site fidelity was weak, indicating that successive trips over the winter target different geographic areas. We suggest that over a season, changes in prey availability are predictable enough for individuals to shift foraging area in response, with limited associated energetic costs. Conversely, over multiple years, the availability of prey resources is less spatially and temporally predictable, increasing the potential costs of shifting foraging area and favouring long-term site fidelity. In a dynamic and patchy environment, multi-year foraging site fidelity may confer a long-term energetic advantage to the individual. Such behaviours that operate at the individual level have evolutionary and ecological implications and are potential drivers of niche specialization and modifiers of intra-specific competition.     

Stretch-activated channels in pulmonary arterial smooth muscle cells from normoxic and chronically hypoxic rats

Stretch-activated channels (SACs) act as membrane mechanotransducers since they convert physical forces into biological signals and hence into a cell response. Pulmonary arterial smooth muscle cells (PASMCs) are continuously exposed to mechanical stimulations e.g., compression and stretch, that are enhanced under conditions of pulmonary arterial hypertension (PAH). Using the patch-clamp technique (cell-attached configuration) in PASMCs, we showed that applying graded negative pressures (from 0 to -60 mmHg) to the back end of the patch pipette increases occurrence and activity of SACs. The current-voltage relationship (from -80 to +40 mV) was almost linear with a reversal potential of 1 mV and a slope conductance of 34 pS. SACs were inhibited in the presence of GsMTx-4, a specific SACs blocker. Using microspectrofluorimetry (indo-1), we found that hypotonic-induced cell swelling increases intracellular Ca(2+) concentration ([Ca(2+)](i)). This [Ca(2+)](i) increase was markedly inhibited in the absence of external Ca(2+) or in the presence of the following blockers of SACs: gadolinium, streptomycin, and GsMTx-4. Interestingly, in chronically hypoxic rats, an animal model of PAH, SACs were more active and hypotonic-induced calcium response in PASMCs was significantly higher (nearly a two-fold increase). Moreover, unlike in normoxic rats, intrapulmonary artery rings from hypoxic rats mounted in a Mulvany myograph, exhibited a myogenic tone sensitive to SAC blockers. In conclusion, this work demonstrates that SACs in rat PASMCs can be activated by membrane stretch as well as hypotonic stimulation and are responsible for [Ca(2+)](i) increase. The link between SACs activation-induced calcium response and myogenic tone in chronically hypoxic rats suggests that SACs are an important element for the increased pulmonary vascular tone in PAH and that they may represent a molecular target for PAH treatment.     

Role of the gap junctions in the contractile response to agonists in pulmonary artery from two rat models of pulmonary hypertension

Background

Pulmonary hypertension (PH) is characterized by arterial vascular remodelling and alteration in vascular reactivity. Since gap junctions are formed with proteins named connexins (Cx) and contribute to vasoreactivity, we investigated both expression and role of Cx in the pulmonary arterial vasoreactivity in two rat models of PH.

Methods

Intrapulmonary arteries (IPA) were isolated from normoxic rats (N), rats exposed to chronic hypoxia (CH) or treated with monocrotaline (MCT). RT-PCR, Western Blot and immunofluorescent labelling were used to study the Cx expression. The role of Cx in arterial reactivity was assessed by using isometric contraction and specific gap junction blockers. Contractile responses were induced by agonists already known to be involved in PH, namely serotonin, endothelin-1 and phenylephrine.

Results

Cx 37, 40 and 43 were expressed in all rat models and Cx43 was increased in CH rats. In IPA from N rats only, the contraction to serotonin was decreased after treatment with ^37-43Gap27, a specific Cx-mimetic peptide blocker of Cx 37 and 43. The contraction to endothelin-1 was unchanged after incubation with ⁴⁰Gap27 (a specific blocker of Cx 40) or ^37-43Gap27 in N, CH and MCT rats. In contrast, the contraction to phenylephrine was decreased by ⁴⁰Gap27 or ^37-43Gap27 in CH and MCT rats. Moreover, the contractile sensitivity to high potassium solutions was increased in CH rats and this hypersensitivity was reversed following ^37-43Gap27 incubation.

Conclusion

Altogether, Cx 37, 40 and 43 are differently expressed and involved in the vasoreactivity to various stimuli in IPA from different rat models. These data may help to understand alterations of pulmonary arterial reactivity observed in PH and to improve the development of innovative therapies according to PH aetiology.     

NO-induced modulation of calcium-oscillations in pulmonary vascular smooth muscle

The effect of the nitric oxide (NO) donor sodium nitroprusside (SNP) on both [Ca(2+)](i)and mechanical activity was studied in the rat isolated pulmonary artery (RPA). In freshly isolated myocytes loaded with 1 microM indo-lacetoxymethyl ester for 30 min, short (40-60 s) application of ATP (100 microM) or ET-1 (0.1 microM) induced 3-6 cyclic rises in [Ca(2+)](i)(Ca-oscillations) of decreasing amplitude. Preincubation of cells with SNP (10-250 microM) for 10 min had no effect on the resting [Ca(2+)](i)value, but progressively abolished the oscillations. A similar effect was obtained with 8-bromo-cGMP (100-500 microM). SNP (0.001-100 microM) concentration-dependently relaxed ATP (10 mM, n = 4) and ET-1 (0.1 microM, n = 4)-precontracted RPA. 1H-[1,2,4]oxadiazolol [4,3,-a]quinoxalin-1-one (ODQ, 10 microM), a potent inhibitor of the cytosolic guanylyl cyclase, fully reversed the effect of SNP on ATP-induced [Ca(2+)](i)oscillations as well as on ATP-precontracted RPA. In contrast, N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H8, 10 microM), a potent inhibitor of cGMP-dependent protein kinase (PKG), did not alter the effect of SNP. Caffeine (5 mM) induced only one transient [Ca(2+)](i)-increase (n = 24), the amplitude of which was altered neither by SNP nor by 8-bromo-cGMP. Our results show that the relaxing effect of NO in RPA is related, at least in part, to its action on the Ca-signalling pathway. NO interacts with inositol trisphosphate pathway without interacting with the ryanodine-sensitive receptor. Finally, the effect of NO involves an increase in cGMP but appears independent of activation of PKG.     

Fraktalkine produced by airway smooth muscle cells contributes to mast cell recruitment in asthma

Human airway smooth muscle cells (HASMC) secrete fractalkine (FKN), a chemokine the concentration of which is increased in asthmatic patients. HASMC also induce mast cell chemotaxis, as a component of asthma inflammation. We therefore evaluated the role of smooth muscle-derived FKN in mast cell migration. We assessed the capacity of recombinant FKN to induce human mast cell chemotaxis. This effect implicates a calcium-independent pathway involving actin reorganization and protein kinase C-delta. We found that HASMC constitutively produce FKN, the synthesis of which is reinforced upon proinflammatory stimulation. Under basal experimental conditions, FKN production by HASMC is not sufficient to induce mast cell chemotaxis. However, pretreatment of mast cells with the neuropeptide vasoactive intestinal peptide (VIP) increases FKN potency to attract mast cells. Since we observed, in asthmatic patients, an increase in both FKN and VIP expression by airway smooth muscle and a positive correlation between VIP staining and mast cell infiltration of the smooth muscle layer, we conclude that HASMC-derived FKN may contribute to mast cell recruitment in asthma.     

Serotonin-induced activation of TRPV4-like current in rat intrapulmonary arterial smooth muscle cells

In the present study, we investigated the implication of transient receptor potential vanilloid (TRPV)-related channels in the 5-hydroxytryptamine (5-HT)-induced both intracellular calcium response and mitogenic effect in rat pulmonary arterial smooth muscle cells (PASMC). Using microspectrofluorimetry (indo-1 as Ca(2+) fluorescent probe) and the patch-clamp technique (in whole-cell configuration), we found that 5-HT (10 microM) induced a transient intracellular calcium mobilization followed by a sustained calcium entry. This latter was partly blocked by an inhibitor of cytochrome P450 epoxygenase (17-ODYA) and insensitive to cyclo-oxygenase and lipoxygenase inhibitors (indomethacin and CDC), suggesting the involvement of arachidonic acid metabolization by cytochrome P450 epoxygenase. This calcium influx was also sensitive to Ni(2+) and to ruthenium red, a TRPV channel blocker, and mimicked by 4alpha-phorbol-12,13-didecanoate (4alpha-PDD), a TRPV4 channel agonist. In patched PASMC, 5-HT and 4alpha-PDD-activated TRPV4-like ruthenium red sensitive currents with typical characteristics. Furthermore, 5-HT induced a ruthenium red sensitive increase in BrdU incorporation levels in PASMC. The present study provides evidence that 5-HT activates a TRPV4-like current, potentially involved in PASMC proliferation. The signalling pathway between proliferation and ion channel activation remains to be determined and may represent a molecular target for the treatment of vascular diseases such as pulmonary hypertension.     

Pregnant rat myometrial cells show heterogeneous ryanodine- and caffeine-sensitive calcium stores

IntracellularCa²⁺ release channels such asryanodine receptors play crucial roles in theCa²⁺-mediated signaling thattriggers excitation-contraction coupling in muscles. Although theexistence and the role of these channels are well characterized inskeletal and cardiac muscles, their existence in smooth muscles, andmore particularly in the myometrium, is very controversial. We have nowclearly demonstrated the expression of ryanodine receptorCa²⁺ release channels in ratmyometrial smooth muscle, and for the first time, intracellularCa²⁺ concentration experimentswith indo 1 on single myometrial cells have revealed the existence of afunctional ryanodine- and caffeine-sensitive Ca²⁺ release mechanism in 30% ofrat myometrial cells. RT-PCR and RNase protection assay on wholemyometrial smooth muscle demonstrate the existence of all threeryr mRNAs in the myometrium:ryr3 mRNA is the predominant subtype,with much lower levels of expression forryr1 andryr2 mRNAs, suggesting that theryanodine Ca²⁺ release mechanismin rat myometrium is largely encoded byryr3. Moreover, using intracellularCa²⁺ concentration measurementsand RNase protection assays, we have demonstrated that the expression,the percentage of cells responding to ryanodine, and the function ofthese channels are not modified during pregnancy.

     

First-year survival of southern elephant seals,Mirounga leonina,at sub-Antarctic Macquarie Island

Juvenile seals branded on the isthmus of Macquarie Island as pups displayed a high degree of philopatry. They returned more often and in greater densities to the northern third of the island within 10 km of their birth sites. Juvenile seals were observed to haul out more frequently and in greater numbers on the east coast as opposed to the west. Juvenile seals typically hauled out on two occasions, once during the winter, and once to moult. The probability of recapturing (resighting) branded and tagged seals was greater during the mid-year haulout. First-year survival estimates were obtained from searches of all Macquarie Island beaches for marked (branded and tagged) seals. From a branded population of 2000 seals, 897 were known to be alive at age 1 year, and minimum first-year survival was calculated at 44.85%. To this minimum estimate was added the number of seals overlooked during systematic and standardised searches of the island, and a revised estimate of 65.60% was calculated. Survival rates calculated using a custom model and a conventional mark-recapture model (MARK) were compared and no differences detected. Actual survival data and probability of sighting estimates were included in the revised estimate of first-year survival of southern elephant seals at Macquarie Island. There were no differences in the number of surviving males and females. Accepted: 25 October 1998