Birte Juul

MECHANISMS OF CA2+ SENSITIZATION OF FORCE PRODUCTION BY NORADRENALINE IN RAT MESENTERIC SMALL ARTERIES

1 Mechanisms of Ca2+ sensitization of force production by noradrenaline were investigated by measuring contractile responses, intracellular Ca2+ concentration ([Ca2+]i) and phosphorylation of the myosin light chain (MLC) in intact and α‐toxin‐permeabilized rat mesenteric small arteries. 2 The effects of noradrenaline were investigated at constant membrane potential by comparing fully depolarized intact arteries in the absence and presence of noradrenaline. Contractile responses to K‐PSS (125 mM K+) and NA‐K‐PSS (K‐PSS + 10 μM noradrenaline) were titrated to 30 and 75 %, respectively, of control force, by adjusting extracellular Ca2+ ([Ca2+]o). At both force levels, [Ca2+]i was substantially lower with NA‐K‐PSS than with K‐PSS. With K‐PSS, the proportion of MLC phosphorylated (≈30 %) was similar at 30 and 75 % of control force; with NA‐K‐PSS, MLC phosphorylation was greater at the higher force level (40 vs. 34 %). 3 In α‐toxin‐permeabilized arteries, the force response to 1 μM Ca2+ was increased by 10 μM noradrenaline, and MLC phosphorylation was increased from 35 to 45 %. The protein kinase C (PKC) inhibitor calphostin C (100 nM) abolished the noradrenaline‐induced increase in MLC phosphorylation and contractile response, without affecting the contraction in response to Ca2+. Treatment with ATPγS in the presence of the MLC kinase inhibitor ML‐9 increased the sensitivity to Ca2+ and abolished the response to noradrenaline. 4 The present results show that in rat mesenteric small arteries noradrenaline‐induced Ca2+ sensitization is associated with an increased proportion of phosphorylated MLC. The results are consistent with a decreased MLC phosphatase activity mediated through PKC. Furthermore, while MLC phosphorylation is a requirement for force production, the results show that other factors are also involved in force regulation.

Responses of femoral resistance vessels to angiotensin in vitro

The effect of angiotensin II and angiotensin I on isolated rat resistance vessels (inner diameter ca. 200 micron) was investigated. Angiotensin II caused a contraction (ED50 = 0.58 +/- 0.17 X 10(-8) M) of rat femoral and cerebral arteries and to a lesser extent of mesenteric and renal arteries. However, all vessels showed strong tachyphylaxis on repeated stimulation with angiotensin II. Tachyphylaxis was avoided by inducing submaximal tone in the vessels with either K, noradrenaline or serotonin. The response to angiotensin II was inhibited by saralasin but not by captopril. Angiotensin I also caused contraction of the femoral arteries (ED50 = 2.68 +/- 0.32 X 10(-8) M). These responses were inhibited by captopril and saralasin. Functional removal of the endothelium had little effect on the contractile responses to either angiotensin I or II. These results indicate that there are functional receptors to angiotensin II in the resistance vessels of the rat and that, in the presence of tone (a more physiological condition), the vessels contract to angiotensin II without tachyphylaxis. In addition, angiotensin II may be formed from angiotensin I by the angiotensin converting enzyme which may be situated in the vessel wall as well as in the endothelium.

Probing of the Membrane Topology of Sarcoplasmic Reticulum Ca2+-ATPase with Sequence-specific Antibodies EVIDENCE FOR PLASTICITY OF THE C-TERMINAL DOMAIN

The topology of Ca2+-ATPase in sarcoplasmic reticulum (SR) vesicles was investigated with the aid of sequence-specific antibodies, produced against oligopeptides corresponding to sequences close to the membranous portions of the protein. The antisera in competitive enzyme-linked immunosorbent assays only reacted with intact SR vesicles to a limited extent, but most epitopic regions were exposed by low concentrations of nondenaturing detergent, octaethylene glycol dodecyl ether (C12E8) or after removal of cytosolic regions by proteinase K. In particular, these treatments exposed the loop regions in the C-terminal domain, including L7–8, the loop region located between transmembrane segments M7 and M8, with a putative intravesicular position, which had immunochemical properties very similar to those of the C terminus with a documented cytosolic exposure. In contrast to this, the reactivity of the N-terminal intravesicular loop regions L1–2 and L3–4 was only increased by C12E8 treatment but not by proteinase K proteolysis. Complexation of Ca2+-ATPase with β,γ-CrATP stabilized the C-terminal domain of Ca2+-ATPase against proteinase K proteolysis and reaction with most of the antisera, but immunoreactivity was maintained by the L6–7 and L7–8 loops. Immunoelectron microscopic analyses of vesicles following negative staining, thin sectioning, and the SDS-digested freeze-fracture labeling method suggested that the L7–8 epitope, in contrast to L6–7 and the C terminus, can be exposed on either the intravesicular or cytosolic side of the membrane. A preponderant intravesicular location of L7–8 in intact vesicles is suggested by the susceptibility of this region to proteolytic cleavage after disruption of the vesicular barrier with C12E8 and in symmetrically reconstituted Ca2+-ATPase proteoliposomes. In conclusion, our data suggest an adaptable membrane insertion of the C-terminal Ca2+-ATPase domain, which under some conditions permits sliding of M8 through the membrane with cytosolic exposure of L7–8, of possible functional significance in connection with Ca2+translocation. On the technical side, our data emphasize that extreme caution is needed when using nondenaturing detergents or other treatments like EGTA at alkaline pH to open up vesicles for probing of intravesicular location with antibodies.

Nucleotide hydrolytic activity of isolated intact rat mesenteric small arteries

Segments of isolated intact rat mesenteric small arteries were incubated in physiological bicarbonate buffer in the presence of nano- to millimolar concentrations of ATP. ATP was hydrolysed, and when the vessel was transferred from one incubation to another, the enzyme activity was transferred with the vessel, consistent with the presence of an ecto-ATPase. The substrate, ATP, was shown to induce a modification of the hydrolytic activity which occurred the more rapidly the higher the concentration of ATP. The modified system hydrolysed ATP with a decreased substrate affinity. As the substrate induced a modification of the hydrolytic activity, steady-state velocity measurements for determination of kinetic parameters could not be obtained. Nevertheless, it was possible to compare the modification caused by ATP and UTP, and to compare the hydrolysis rates measured with [32P]ATP, [32P]UTP and [32P]GTP. It was concluded that the hydrolytic activity of the vessels did not distinguish between the nucleoside triphosphates (NTPs). In a histidine buffer, the activity was shown to be activated by micromolar concentrations of either Ca2+ or Mg2+, and not to be influenced by inhibitors of P-type, F-type and V-type ATPases. Functional removal of the endothelium before assay did not reduce the measured NTP hydrolysis. At millimolar concentrations of trinucleotide the hydrolysis rate was 10-15 mumol per min per gram of tissue or 0.11-0.17 mumol per min per 10(6) vascular smooth muscle cells. This value is equivalent to the maximal velocity obtained for the Ca2+ or Mg(2+)-dependent NTPase released to the medium upon 2 s of sonication of the vessels (Plesner, L., Juul, B., Skriver, E. and Aalkjaer, C. (1991) Biochim. Biophys. Acta 1067, 191-200). Comparing the characteristics of the released NTPase to the characteristics of the activity of the intact vessel, they showed a strong resemblance, but the substrate-induced modification of the enzyme was seen only in the intact preparation.

Contractile Effects of Tetradecapeptide Renin Substrate on Rat Femoral Resistance Vessels

The effect of products of the renin-angiotensin system on the contractile response of rat resistance vessels (internal diameter ˜200 μm) has been investigated. The vessels were isolated from the femoral bed and segments of the vessels were mounted on an isometric myograph. The vessels responded in a concentration-dependent manner to synthetic tetradecapeptide (TDP) renin substrate, angiotensin I (ANG I) and angiotensin II (ANG II), the responses to all these substances being inhibited by saralasin (0.1 μ mol/l). The responses to ANG I, but not those to TDP renin substrate, were inhibited by captopril (1 mmol/l). In contrast, the non-specific protease inhibitor aprotinin had an inhibitory effect on responses to TDP renin substrate. The results suggest that TDP renin substrate is converted to ANG II by a process that does not involve metabolism of ANG I.

Proteolytic Studies on the Transduction Mechanism of Sarcoplasmic Reticulum Ca2+-ATPase

Abstract: After proteinase K‐induced excision of five amino acid residues in the semiconserved polypeptide chain linking the end of the A domain with the S3/M3 transmembrane segment we find that Ca2+ transport is blocked while partial reactions like Ca2+ binding, ATP phosphorylation, and Ca2+‐occlusion are left intact. However, formation of the so‐called E2P state (either from the phosphorylated species formed in the presence of ATP and Ca2+ or from the Ca2+‐depleted unphosphorylated species) is blocked. We conclude that the proteinase K‐treated ATPase, while maintaining many of the partial reactions, is incapable of energy transduction because of the absence of an E2P state with Ca2+ binding sites exposed to the intravesicular space. Sequence comparisons and mutagenesis data point to an important role in energy transduction of P‐type ATPases of a conserved motif located at the end of the A domain.

The Topology of Sarcoplasmic Reticulum Ca2+-ATPase and Na+,K+-ATPase in the M5/M6 Region

While there is general agreement concerning the existence of 4 hydrophobic membrane traverses in the N-terminal part of P-type ATPases, the exact topology of the C-terminal, membraneous domain is still a matter of dispute. For sarcoplasmic reticulum (SR) Ca2+-ATPase 3 pairs of transmembrane helices (M5-M10) were proposed, leading to a 10-helical model for this ATPase, cf. Fig. 1. For Na+,K+-ATPase fewer membrane traverses were considered probable, leading to 7 helical (8) or 8 helical (3) models. In the 7 helical model, regions corresponding to M6, M8, and M10 were placed outside the lipid membrane, based on protein-chemical and immunochemical evidence. However, since there is strong evidence for cytosolic exposure of both the N-terminus and C-terminus, 8 helical models are now favored for Na+,K+-ATPase, but exactly how the transmembrane segments would be positioned in the membrane remains undefined. Concerning the different models proposed for Ca2+-ATPase and Na+,K+-ATPase it also needs to be asked, if it is plausible that their topology should be different, considering the fact that the hydropathic profiles of the two enzymes are strikingly similar.