Juan A. Rosado

Coupling between inositol 1,4,5-trisphosphate receptors and human transient receptor potential channel 1 when intracellular Ca2+ stores are depleted

In the present study we have investigated the role of inositol 1,4, 5-trisphosphate (IP(3)), functional IP(3) receptors (IP(3)Rs) and the human homologue of the Drosophila transient receptor potential (Trp) channel, human Trp1 (hTrp1), in store-mediated Ca(2+) entry (SMCE) in human platelets. Inhibition of IP(3) recycling using Li(+), or the inhibition of IP(3)Rs using xestospongin C, both resulted in the inhibition of SMCE activation following Ca(2+) store depletion using thapsigargin. Co-immunoprecipitation experiments indicated that endogenously expressed hTrp1 couples with IP(3)R type II, but not types I or III, in platelets with depleted intracellular Ca(2+) stores, but not in control, undepleted cells. These results provide strong evidence for the activation of SMCE by conformational coupling involving de novo association between IP(3)Rs and a plasma membrane channel in normal human cells.

Endogenously Expressed Trp1 Is Involved in Store-mediated Ca2+ Entry by Conformational Coupling in Human Platelets

Physical interaction between transient receptor potential (Trp) channels and inositol 1,4,5-trisphosphate receptors (IP3Rs) has been presented as a candidate mechanism for the activation of store-mediated Ca2+ entry. The role of a human homologue of Drosophila transient receptor potential channel, hTrp1, in the conduction of store-mediated Ca2+ entry was examined in human platelets. Incubation of platelets with a specific antibody, which recognizes the extracellular amino acid sequence 557–571 of hTrp1, inhibited both store depletion-induced Ca2+ and Mn2+ entry in a concentration-dependent manner. Stimulation of platelets with the physiological agonist thrombin activated coupling between the IP3 receptor type II and endogenously expressed hTrp1. This event was reversed by refilling of the internal Ca2+ stores but maintained after removal of the agonist if the stores were not allowed to refill. Inhibition of IP3 recycling using Li+ or inhibition of IP3Rs with xestospongin C or treatment with jasplakinolide, to stabilize the cortical actin filament network, abolished thrombin-induced coupling between hTrp1 and IP3R type II. Incubation with the anti-hTrp1 antibody inhibited thrombin-evoked Ca2+ entry without affecting Ca2+ release from intracellular stores. These results provide evidence for the involvement of hTrp1 in the activation of store-mediated Ca2+ entry by coupling to IP3R type II in normal human cells.

The actin cytoskeleton in store-mediated calcium entry

Store‐mediated Ca2+ entry is the main pathway for Ca2+ influx in platelets and many other cells. Several hypotheses have considered both direct and indirect coupling mechanisms between the endoplasmic reticulum and the plasma membrane. Here we pay particular attention to new insights into the regulation of store‐mediated Ca2+ entry: the role of the cytoskeleton in a secretion‐like coupling model. In this model, Ca2+ entry may be mediated by a reversible trafficking and coupling of the endoplasmic reticulum with the plasma membrane, that shows close parallels to the events mediating secretion. As with secretion, the actin cytoskeleton plays an inhibitory role in the activation of Ca2+ entry by preventing the approach and coupling of the endoplasmic reticulum with the plasma membrane, making cytoskeletal remodelling a key event in the activation of Ca2+ entry. We also review recent advances investigating the regulation of store‐mediated Ca2+ entry by small GTPases and phosphoinositides, which might be involved in the store‐mediated Ca2+ entry pathway through roles in the remodelling of the cytoskeleton.

A role for the actin cytoskeleton in the initiation and maintenance of store-mediated calcium entry in human platelets.

Store-mediated Ca(2+) entry (SMCE) is a major pathway for Ca(2+) influx in many cells, yet how depletion of the intracellular Ca(2+) stores leads to the activation of Ca(2+) entry across the plasma membrane is not well understood. Recent work in platelets favors a secretion-like conformational coupling mechanism involving proteins in the plasma membrane (PM) and in the membrane of the Ca(2+) store, located in the endoplasmic reticulum (ER). The activation and maintenance of SMCE in platelets has been shown to depend on remodeling of the actin cytoskeleton, which may be required to allow trafficking of the ER toward the PM to permit coupling to occur and to stabilize this coupling once achieved. The coupling itself has been shown to involve one isoform of the inositol 1,4,5-trisphosphate receptor (IP(3)RII) and the Ca(2+)-permeable channel protein, human Trp1 (hTrp1).

Activation of store-mediated calcium entry by secretion-like coupling between the inositol 1,4,5-trisphosphate receptor type II and human transient receptor potential (hTrp1) channels in human platelets.

Physical coupling between inositol 1,4,5-trisphosphate (IP(3)) receptors and transient receptor potential (Trp) channels has been demonstrated in both transfected and normal cells as a candidate mechanism for the activation of store-mediated Ca(2+) entry (SMCE). We have investigated the properties of the coupling between the type II IP(3) receptor and naturally expressed human Trp1 (hTrp1) in human platelets. Treatment with xestospongin C, an inhibitor of IP(3) receptor function, abolished SMCE and coupling between the IP(3) receptor and hTrp1. The coupling was activated by depletion of the intracellular Ca(2+) stores, and was reversed by refilling of the stores. We have also examined the role of actin filaments in the activation and maintenance of the coupling. Stabilization of the cortical actin network with jasplakinolide prevented the coupling, indicating that, as with secretion, the actin filaments at the cell periphery act as a negative clamp which prevents constitutive coupling. In addition, the actin cytoskeleton plays a positive role, since disruption of the actin network inhibited the coupling when the Ca(2+) stores were depleted. These results provide strong evidence for the activation of SMCE by a secretion-like coupling mechanism involving a reversible association between IP(3) receptors and hTrp1 in normal human cells.

Farnesylcysteine analogues inhibit store-regulated Ca2+ entry in human platelets: evidence for involvement of small GTP-binding proteins and actin cytoskeleton.

We have investigated the mechanism of Ca(2+) entry into fura-2-loaded human platelets by preventing the prenylation of proteins such as small GTP-binding proteins. The farnesylcysteine analogues farnesylthioacetic acid (FTA) and N-acetyl-S-geranylgeranyl-L-cysteine (AGGC), which are inhibitors of the methylation of prenylated and geranylgeranylated proteins respectively, significantly decreased thrombin-evoked increases in intracellular free Ca(2+) concentration ([Ca(2+)](i)) in the presence, but not in the absence, of external Ca(2+), suggesting a relatively selective inhibition of Ca(2+) entry over internal release. Both these compounds and N-acetyl-S-farnesyl-L-cysteine, which had similar effects to those of FTA, also decreased Ca(2+) entry evoked by the depletion of intracellular Ca(2+) stores with thapsigargin. The inactive control N-acetyl-S-geranyl-L-cysteine was without effect. Patulin, an inhibitor of prenylation that is inert with respect to methyltransferases, also decreased store-regulated Ca(2+) entry. Cytochalasin D, an inhibitor of actin polymerization, significantly decreased store-regulated Ca(2+) entry in a time-dependent manner. Both cytochalasin D and the farnesylcysteine analogues FTA and AGGC inhibited actin polymerization; however, when evoking the same extent of decrease in actin filament formation, FTA and AGGC showed greater inhibitory effects on Ca(2+) entry, indicating a cytoskeleton-independent component in the regulation of Ca(2+) entry by small GTP-binding-protein. These findings suggest that prenylated proteins such as small GTP-binding proteins are involved in store-regulated Ca(2+) entry through actin cytoskeleton-dependent and cytoskeleton-independent mechanisms in human platelets.

Protein kinase C activates non‐capacitative calcium entry in human platelets

1 In many non‐excitable cells Ca2+ influx is mainly controlled by the filling state of the intracellular Ca2+ stores. It has been suggested that this store‐mediated or capacitative Ca2+ entry is brought about by a physical and reversible coupling of the endoplasmic reticulum with the plasma membrane. Here we provide evidence for an additional, non‐capacitative Ca2+ entry mechanism in human platelets. 2 Changes in cytosolic Ca2+ and Sr2+ were measured in human platelets loaded with the fluorescent indicator fura‐2. 3 Depletion of the internal Ca2+ stores with thapsigargin plus a low concentration of ionomycin stimulated store‐mediated cation entry, as demonstrated upon Ca2+ or Sr2+ addition. Subsequent treatment with thrombin stimulated further divalent cation entry in a concentration‐dependent manner. 4 Direct activation of protein kinase C (PKC) by phorbol‐12‐myristate‐13‐acetate or 1‐oleoyl‐2‐acetyl‐sn‐glycerol also stimulated divalent cation entry, without evoking the release of Ca2+ from intracellular stores. Cation entry evoked by thrombin or activators of PKC was abolished by the PKC inhibitor Ro‐31‐8220. 5 Unlike store‐mediated Ca2+ entry, jasplakinolide, which reorganises actin filaments into a tight cortical layer adjacent to the plasma membrane, did not inhibit divalent cation influx evoked by thrombin when applied after Ca2+ store depletion, or by activators of PKC. Thrombin also activated Ca2+ entry in platelets in which the release from intracellular stores and store‐mediated Ca2+ entry were blocked by xestospongin C. These results indicate that the non‐capacitative divalent cation entry pathway is regulated independently of store‐mediated entry and does not require coupling of the endoplasmic reticulum and the plasma membrane. 6 These results support the existence of a mechanism for receptor‐evoked Ca2+ entry in human platelets that is independent of Ca2+ store depletion. This Ca2+ entry mechanism may be activated by occupation of G‐protein‐coupled receptors, which activate PKC, or by direct activation of PKC, thus generating non‐capacitative Ca2+ entry alongside that evoked following the release of Ca2+ from the intracellular stores.

The ERK Cascade, a New Pathway Involved in the Activation of Store-Mediated Calcium Entry in Human Platelets☆

Store-mediated Ca(2+) entry (SMCE) is the main pathway for Ca(2+) influx in platelets and other nonexcitable cells, yet how depletion of the intracellular Ca(2+) stores leads to the activation of Ca(2+) entry across the plasma membrane remains unclear. Recent work in platelets favors a secretionlike conformational coupling mechanism involving the Ca(2+)-permeable channel protein, Trp1, in the plasma membrane and the type-II inositol 1,4,5-trisphosphate receptor in the membrane of the Ca(2+) store, which is located in the endoplasmic reticulum. Extracellular signal-regulated kinases (ERKs) are common participants in a broad variety of signal transduction pathways in human platelets, and inactivation of the ERK cascade has been shown to reduce Ca(2+) entry stimulated by thapsigargin or thrombin. The role of ERK in SMCE into human platelets was found to be independent of the cytoskeleton and a downstream effector of the small guanosine-triphosphate-binding protein Ras.

Dendroaspis natriuretic peptide-like immunoreactivity and its regulation in rat aortic vascular smooth muscle.

Dendroaspis natriuretic peptide (DNP) is a recently isolated 38 amino acid peptide that shares structural and functional properties with the other members of the natriuretic peptide family. The present study demonstrates the presence of DNP-like immunoreactivity in sections of rat aorta, carotid artery and renal vasculature and tubules. DNP-like immunoreactivity was detected in culture aortic vascular smooth muscle cells and medium and is regulated by endothelin-1, angiotensin II and sodium nitroprusside but not by transforming growth factor-beta. Our observations indicate that DNP elicits a marked inhibitory effect on DNA synthesis in culture rat aortic vascular smooth muscle cells.

A-type natriuretic peptide receptor in the spontaneously hypertensive rat kidney

Renal NPR-A binding characteristics was examined in SHR. Renal ANP binding sites of NPR-A showed a lower maximal binding capacity and higher affinity in SHR than in WKY at all intrarenal sites. Despite the lower B(max) in SHR, both ANP(1-28) and ANP(5-25) stimulate similar or greater cGMP production in isolated glomeruli. Studies on guanylate cyclase from glomerular and papillary membranes have reported an increased basal and stimulated guanylate cyclase activity in SHR. The present study provides further evidences for altered NPR-A receptors in SHR kidney, which might act as a negative feedback in response to hypertension.