Characterisation of Ca2+ or Mg(2+)-dependent nucleoside triphosphatase from rat mesenteric small arteries.

When isolated rat mesenteric small arteries were submitted to 2 s of sonication, a nucleoside triphosphatase activity was released to the medium, mainly from the plasma membrane of the vascular smooth muscle cells. The activity was kinetically characterized: It hydrolysed ATP, UTP and GTP with the same substrate affinity and the same specific activity. CaATP, as well as MgATP were substrates for the enzyme with an apparent Km in the micromolar range. ATPase inhibitors: ouabain, vanadate, AlF4-, oligomycin and N-ethylmaleimide were without effect on the hydrolytic activity. Among other modifiers tested only N,N'-dicyclohexylcarbodiimide caused significant (greater than 30%) inhibition. In the presence of micromolecular concentrations of Ca2+ and Mg2+, small (less than 20 mM) concentrations of Na+, K+, Rb+, Cs+ and choline+, irrespective of the nature of the anion, activated the hydrolysis with an equilibrium ordered pattern, but concentrations of monovalent cation salts above 20 mM decreased the hydrolysis rate. No activation by monovalent cation salts was seen at millimolar concentrations of divalent cations and substrate. On the basis of the results a standard mixture is proposed, which allows a sensitive assay of the specific enzyme activity.

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.

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.

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 approximately 200 microns) 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 mumol/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.

Synthetic atrial natriuretic factor is a specific dilator of noradrenaline and serotonin activated renal resistance vessels.

The action of a synthetic 'atrial natriuretic factor' (sANF) on induced tone in isolated rat renal resistance vessels (lumen diameter about 200 microns) was examined and compared with the effects of sANF on resistance vessels of similar size taken from the cerebral, mesenteric and femoral vasculature. Synthetic ANF caused a relaxation of the renal vessels when these were submaximally activated with noradrenaline or serotonin, but had no effect on the responses of the other vessels to these agonists. In contrast to previous reports concerning rabbit aortic vessels, methylene blue (which is thought to cause inhibition of guanyl cyclase) did not reduce the dilator response to sANF in the renal vessels. The results demonstrate that sANF has a specific relaxing effect on renal resistance vessels, and are consistent with its effect being mediated through specific receptors. The mechanism of this relaxant effect remains unknown.

Effects of ATP and related nucleotides on the tone of isolated rat mesenteric resistance arteries.

The relationship between ecto-ATPase activity and the vasoactive effect of ATP is unclear. Previously we have characterized the ectonucleoside triphosphatase activity of isolated rat mesenteric small arteries and now characterize the effect of nucleotides on the tone of these arteries. In resting arteries, ATP caused concentration-dependent contractions that were transient and could not be reproduced within 2 h. Transient contractions in response to ATP also were elicited in arteries precontracted with norepinephrine, but the potency of ATP was increased and responses to repeated stimulations could be obtained. Contractions were followed by relaxation. The response to ATP was unaffected by 100 microM theophylline, 1 microM propranolol or removal of the endothelium. Transient contractions followed by relaxation were caused also by ADP, 2-methyl-thio-ATP (2meSATP) and alpha, beta-methylene-ATP (alpha, beta-meATP). UTP caused sustained contractions, whereas GTP and ITP had little effect. The rank order of potency (alpha, beta-,mATP > ATP > ADP) suggested that P2x purinoceptors were responsible for the contractions, whereas the rank order of potency for the relaxation (alpha, beta-meATP > or = ATP > 2meSATP) was not consistent with the relaxation being mediated by P2Y purinoceptors as defined originally. Desensitization of the contractile response to ATP by alpha, beta-meATP was variable. In contrast, inhibition of the response to ATP was obtained consistently and dose-dependently with GTP.

Effects of ATP and UTP on [Ca2+]i, membrane potential and force in isolated rat small arteries.

We have investigated excitation-contraction coupling mechanisms associated with the activation of purinoceptors and putative pyrimidinoceptors by assessing the effects of ATP and UTP on cytoplasmic Ca2+ activity ([Ca2+]i), membrane potential (Em) and force in rat mesenteric small arteries. UTP induced a sustained concentration-dependent contractions, closely associated with concentration-dependent increases in [Ca2+]i. Superfusion with 0.1 mM UTP caused a sustained depolarisation of 12 +/- 1 mV (SE, n = 8). In Ca(2+)-free medium, the increase in [Ca2+]i and the contraction obtained with UTP (1 mM) were both transient and were inhibited by prior exposure to noradrenaline (NA). In vessels depolarised with KCl, UTP caused no change in Em, but a sustained increase in force and a transient increase in [Ca2+]i were induced, leading to an increased force/[Ca2+]i ratio. Similar effects on [Ca2+]i, Em and force were observed with ATP; but the effect of ATP on force was transient, whereas the effect on [Ca2+]i and Em declined only slowly. There was no crosstachyphylaxis between the responses to ATP and UTP: in the presence of 1 mM of either, the other drug induced contractions in low concentrations, as if they acted through distinct receptors. The results suggest that both UTP and probably ATP release intracellular Ca2+, possibly from the stores emptied by NA. The sustained response to UTP appears to be due to an influx of extracellular Ca2+. UTP but not ATP was found to enhance the force-generating effect of [Ca2+]i.

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 alpha-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 microM 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 (approximately 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 alpha-toxin-permeabilized arteries, the force response to 1 microM Ca2+ was increased by 10 microM 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 ATP gamma S 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 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.

Do transmembrane segments in proteolyzed sarcoplasmic reticulum Ca(2+)-ATPase retain their functional Ca2+ binding properties after removal of cytoplasmic fragments by proteinase K?

The present study was undertaken to investigate the Ca2+ binding properties of sarcoplasmic reticulum Ca(2+)-ATPase after removal of the cytoplasmic regions by treatment with proteinase K. One of the proteolysis cleavage sites (at the end of M6) was found unexpectedly close to the predicted membrane-water interphase, but otherwise the cleavage pattern was consistent with the presence of 10 transmembrane ATPase segments. C-terminal membranous peptides containing the putative transmembrane segments M7 to M10 accumulated after prolonged proteolysis, as well as large water-soluble fragments containing most of the phosphorylation and ATP-binding domain. Ca2+ binding was intact after cleavage of the polypeptide chain in the N-terminal region, but cuts at other locations disrupted the high affinity binding and sequential dissociation properties characteristic of native sarcoplasmic reticulum, leaving the translocation sites with only weak affinity for Ca2+. High affinity Ca2+ binding could only be maintained when proteolysis and subsequent manipulations took place in the presence of a Ca2+ concentration high enough to ensure permanent occupation of the binding sites with Ca2+. We conclude that in the absence of Ca2+, the complex of membrane-spanning segments in proteolyzed Ca(2+)-ATPase is labile, probably because of relatively free movement or rearrangement of individual segments. Our study, which is discussed in relation to results obtained on Na+,K(+)-ATPase and H+,K(+)-ATPase, emphasizes the importance of the cytosolic segments of the main polypeptide chain in exerting constraints on the intramembranous domain of a P-type ATPase.

Estrogen receptors in the human male bladder, prostatic urethra, and prostate. An immunohistochemical and biochemical study.

The distribution and quantity of estrogen receptors (ERs) in the human male bladder, prostatic urethra and the prostate were studied in eight males with recurrent papillomas of the bladder or monosymptomatic hematuria (median age 61 years), 14 men undergoing transurethral resection due to benign prostatic hyperplasia (median age 70 years), and nine men undergoing cystectomy due to malignant tumour of the bladder (median age 70 years). In the first group of patients, biopsies for immunohistochemical examination were obtained from the bladder vault, bottom, both side-walls, the trigone area, and the mid-portion of the prostatic urethra, and in the second group from three locations of the prostatic urethra (bladder neck, mid-portion and veramontanum). In the third group, tissue specimens were taken from the vault of the bladder, prostatic urethra, and the prostate, for immunohistochemical as well as biochemical analysis. In the first group, ERs were found in three out of eight specimens of the prostatic urethra, and in one of these, ERs were confined to periurethral glands. ERs could not be demonstrated in any of the bladder-biopsies. In the second group, ERs were not found in the bladder neck, but were seen in four preparations from the veramontanum and in two from the midportion of the urethra. ERs were located in the urothelium and periurethral glands. In the third group, ERs were seen immunohistochemically in the prostatic urethra (two cases) and the prostatic stromal tissue (two cases). ERs could be demonstrated in the bladder neither by immunohistochemistry nor biochemically.(ABSTRACT TRUNCATED AT 250 WORDS)

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 beta,gamma-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.

Characterization of a protease-resistant domain of the cytosolic portion of sarcoplasmic reticulum Ca2+-ATPase. Nucleotide- and metal-binding sites.

Treatment of rabbit sarcoplasmic reticulum Ca2+-ATPase with a variety of proteases, including elastase, proteinase K, and endoproteinases Asp-N and Glu-C, results in accumulation of soluble fragments starting close to the ATPase phosphorylation site Asp351 and ending in the Lys605-Arg615 region, well before the conserved sequences generally described as constituting the "hinge" region of this P-type ATPase (residues 670-760). These fragments, designated as p29/30, presumably originate from a relatively compact domain of the cytoplasmic head of the ATPase. They retain two structural characteristics of intact Ca2+-ATPase as follows: high sensitivity of peptidic bond Arg505-Ala506 to trypsin cleavage, and high reactivity of lysine residue Lys515 toward the fluorescent label fluorescein 5'-isothiocyanate. Regarding functional properties, these fragments retain the ability to bind nucleotides, although with reduced affinity compared with intact Ca2+-ATPase. The fragments also bind Nd3+ ions, leaving open the possibility that these fragments could contain the metal-binding site(s) responsible for the inhibitory effect of lanthanide ions on ATPase activity. The p29/30 soluble domain, like similar proteolytic fragments that can be obtained from other P-type ATPases, may be useful for obtaining three-dimensional structural information on the cytosolic portion of these ATPases, with or without bound nucleotides. From our findings we infer that a real hinge region with conformational flexibility is located at the C-terminal boundary of p29/30 (rather than in the conserved region of residues 670-760); we also propose that the ATP-binding cleft is mainly located within the p29/30 domain, with the phosphorylation site strategically located at the N-terminal border of this domain.