Over-expression of NCS-1 in AtT-20 cells affects ACTH secretion and storage.

The effect of over-expressing neuronal calcium sensor 1 (NCS-1) upon stimulated adrenocorticotrophin (ACTH) secretion was studied in AtT-20 cells. Stably-transfected AtT-20 cell lines over-expressing NCS-1 were obtained and compared to wild type AtT-20 cells. Corticotrophin releasing factor (CRF-41)-stimulated ACTH secretion from NCS-1 over-expressing cells was significantly reduced from that obtained in wild type AtT-20 cells. The effects of other stimulants of ACTH secretion from wild type AtT-20 cells were not attenuated in NCS-1 over-expressing cells. Calcium, guanosine 5'-O-(3'-thiotriphosphate) (GTP-gamma-S) and mastoparan stimulated ACTH secretion from permeabilised wild type AtT-20 and NCS-1 over-expressing AtT-20 cells with significantly greater ACTH secretion obtained in NCS-1 over-expressing cells. This study shows that in intact cells over-expression of NCS-1 reduces exocytotic ACTH release, while in permeabilised cells increases ACTH release. NCS-1 has multiple cellular targets and that directly and indirectly via these targets acts to increase the releasable ACTH pool while inhibiting CRF-41 stimulus-secretion coupling.

Effects of phospholipase A(2) activating peptides upon GTP-binding protein-evoked adrenocorticotrophin secretion.

A GTP-binding protein (G-protein), termed G-exocytosis (Ge), mediates the effects of calcium ions in the late stages of the adrenocorticotrophin (ACTH) secretory pathway. An activator of Ge, mastoparan, also stimulates phospholipase A(2) and so a comparison of other phospholipase A(2)-activating peptides, melittin and phospholipase A(2)-activating peptide was made with mastoparan to assess whether phospholipase A(2)activation was an important component of Ge-evoked secretion. All three peptides stimulated ACTH secretion in the effective absence of calcium ions from permeabilised cells, actions potentiated by a phospholipase A(2)inhibitor. Ca(2+)-evoked secretion from permeabilised cells was similarly potentiated by a phospholipase A(2) inhibitor. Furthermore, arachidonic acid inhibited Ca(2+)- and Ge-evoked ACTH secretion, an action blocked by the cyclo-oxygenase inhibitor ibuprofen. This study suggests that the products of phospholipase A(2)-generated arachidonic metabolism may exert an inhibitory action on the late post-Ca(2+) stages of the ACTH secretory pathway and that prostaglandins may be the active agents in this capacity.

Effects of wortmannin upon the late stages of the secretory pathway of AtT-20 cells.

Heterotrimeric GTP-binding (G) proteins, termed Ge, have a role in the late stages of the adrenocorticotrophin (ACTH) secretory pathway in the mouse AtT-20/D16-16 anterior pituitary tumour cell line. The wortmannin sensitivity of Ge-controlled mechanisms in AtT-20 cells was investigated to provide information on the possible mechanisms linking Ge with secretion. Permeabilised cells exposed to calcium ions (10(-9) to 10(-3) M), guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) (10(-8) to 10(-4) M) and mastoparan (10(-8) to 10(-5) M) demonstrated a significant and concentration-dependent stimulation of ACTH secretion from non-stimulated levels for all three agents. Coincubation with wortmannin (10(-5) M) significantly inhibited both calcium-independent and -stimulated secretion. The effect of wortmannin was concentration-dependent being maximal at 10(-6) M. The study shows that wortmannin inhibits both calcium-independent and -stimulated secretion from permeabilised AtT-20 cells indicating a role for phosphatidylinositol-3 kinase in determining the size of the readily releasable pool of ACTH and/or in mediating calcium/Ge-evoked secretion from this pool.

Characterisation of the effects of natriuretic peptides upon ACTH secretion from the mouse pituitary.

The involvement of natriuretic peptides in the regulation of ACTH secretion in mice hemi-pituitary preparations was investigated. Atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) all inhibited CRF (10(-9) M)-evoked ACTH secretion over a concentration range of 10(-12)-10(-10) M and also stimulated cyclic GMP accumulation over a concentration range of 10 (-8)-10(-5) M. CNP was the most effective both in the inhibition of ACTH secretion and in the stimulation of cyclic GMP accumulation. Coincubation of hemi-pituitaries with 8bromo-cyclic GMP (10(-4) M) completely inhibited CRF (10(-9) M)-evoked ACTH secretion. Northern blot analysis revealed that all three major isoforms of the natriuretic peptide receptors are expressed in the mouse pituitary. These results demonstrate that natriuretic peptides do inhibit CRF-stimulated ACTH secretion from mouse pituitary preparations. A role for cGMP in mediating this effect on hormone secretion is indicated but the discrepancy between the efficacies of natriuretic peptides in inhibiting the secretory response and stimulating cyclic GMP accumulation suggest a more complicated stimulus-secretion coupling pathway is in operation.

Heterotrimeric G-protein candidates for Ge in the ACTH secretory pathway.

The mouse AtT-20/D16-16 anterior pituitary tumour cell line was used to identify candidate heterotrimeric G-proteins for G-exocytosis (Ge) which mediates calcium ion-stimulated adrenocorticotrophin (ACTH) secretion in this cell line. AtT-20 cells express several heterotrimeric G-protein alpha subunits; Gs alpha, Gt alpha, Gq alpha, G11alpha, G12alpha, G13alpha, G14alpha, G15alpha, Gz alpha, Gi2alpha, Gi3alpha, and Go alpha and so heterotrimeric G-protein selective agents were used to differentiate between these candidates. Agents which stimulate ACTH secretion via Ge were not pertussis toxin (PTX)-sensitive nor was cholera toxin (CTX) able to stimulate ACTH secretion from permeabilised cells in the absence of calcium. G-protein antagonists which inhibit activation of Gs, Gi, and Gq subfamilies did not attenuate Ge-stimulated ACTH secretion from permeabilised AtT-20 cells. In AtT-20 cells the stimulatory G-protein involved in the late stages of the ACTH secretory pathway does not belong to the Gs, Gi (with the exception of Gz) or Gq subfamilies of heterotrimeric G-proteins leaving Gz, G12 or G13 as the strongest candidates for Ge.

The roles of adenosine 3',5'-cyclic monophosphate-dependent protein kinase A and protein kinase C in stimulus-secretion coupling in AtT-20 cells.

The ACTH-secreting mouse AtT-20/D16-16 anterior pituitary tumour cell line was used to study adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA) and protein kinase C (PKC) involvement in stimulus-secretion coupling pathways. In permeabilised AtT-20 cells under calcium ion-free conditions, forskolin (1O mu M), CRH-41 (1OOnM), guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S; 100 mu M) but not mastoparan (10 mu M) stimulated cAMP accumulation. Measurement of ACTH secretion under identical incubation conditions revealed that GTP-gamma-S and mastoparan significantly stimulated ACTH secretion but forskolin and CRH-41 did not. This dissociates cAMP accumulation from ACTH secretion under calcium ion-free conditions and indicated that the effects of mastoparan and GTP-gamma-S on ACTH secretion are not mediated by cAMP production. Calcium ions (1 nM to I mM) stimulated ACTH secretion from electrically permeabilised cells in a concentration-dependent manner. cAMP (100 mu M) and phorbol 12-myristate 13-acetate (PMA; 100 nM) synergistically enhanced the response to calcium ions. cAMP did not stimulate ACTH secretion in the absence of calcium ions nor did it alter the concentrations at which calcium stimulated ACTH secretion. This suggests that stimulation of ACTH secretion via the calcium-dependent pathway is necessary before any cAMP-mediated enhancement of secretion is manifest. PMA, however, did stimulate ACTH secretion in the absence of calcium ions, indicating distinct mechanisms for PKC-evoked secretion. Co-incubation with cAMP and PMA did not exceed the secretory response obtained with the combination of PMA and calcium ions. CRH-41 (1 pM to 100 nM) and forskolin (1 nM to 100 mu M) stimulated ACTH secretion from intact cells in a concentration-dependent manner. Co-incubation with PMA (100 nM) further enhanced the ACTH response to CRH-41 and forskolin; the effects were simply additive. The present study indicates that there are distinct roles for PKA and PKC in stimulus-secretion coupling in AtT-20 cells. The PKA-dependent pathway, acting in concert with the calcium messenger system, serves as part of the stimulus-secretion coupling pathway by which activation of CRH-41 receptors control ACTH secretion. The PKC-dependent pathway, in contrast, seems to be independent of the calcium messenger system and may represent a separate control mechanism of ACTH secretion.

Effects of mastoparan upon the late stages of the ACTH secretory pathway of AtT-20 cells.

1. The mouse AtT-20/D16-16 anterior pituitary tumour cell line was used as a model system for the study of the effects of mastoparan upon the late stages of the adrenocorticotrophin (ACTH) secretory pathway. 2. Mastoparan (10(-8)-10(-5) M), an activator of heterotrimeric guanosine 5'-triphosphate binding proteins (G-proteins), stimulated ACTH secretion from electrically-permeabilized AtT-20 cells in a concentration-dependent manner in the effective absence of calcium ions with a threshold of 10(-6) M. Guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) (10(-8)-10(-4) M) also stimulated ACTH secretion from electrically-permeabilized AtT-20 cells in a concentration-dependent manner in the effective absence of calcium ions with a threshold of 10(-6) M. This GTP-gamma-S-evoked secretion is consistent with previous studies which demonstrated that a G-protein, termed GE, mediates calcium evoked ACTH secretion from AtT-20 cells. GTP-gamma-S-evoked secretion however was not as great as that obtained in response to mastoparan. 3. Both mastoparan (10(-5) M) and GTP-gamma-S (10(-4) M) stimulated ACTH secretion from electrically-permeabilized AtT20 cells in a time-dependent manner. A time of 30 min was adopted as the standard incubation period for the study of both mastoparan and GTP-gamma-S-stimulated ACTH secretion from permeabilized AtT-20 cells. 4. Mastoparan (10(-8)-10(-5) M) stimulated ACTH secretion from permeabilized AtT-20 cells to the same extent in the presence and absence of the protein kinase C (PKC) inhibitor, chelerythrine chloride (10(-5) M). 5. Mastoparan (10-8 10-5 M)-stimulated ACTH secretion from permeabilized AtT-20 cells was significantly reduced in the presence of guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S, 10-4 M).6. The mastoparan analogue, Mas-7 (10-8-10-5 M) stimulated ACTH secretion from permeabilized AtT-20 cells to a greater extent than mastoparan (10-8 10-5 M) however, the mastoparan analogue Mas-17 (10-8- 10-5 M) had no effect upon ACTH secretion from permeabilized AtT-20 cells.7. Mastoparan (10-8-10-5 M) stimulated ACTH secretion from permeabilized AtT-20 cells in the presence and absence of ATP, normally present in the standard permeabilization medium at a concentration of 5 mM. Mastoparan (10-8- 10-5 M)-stimulated ACTH secretion as well as control secretion was reduced when ATP was omitted.8. The results of the present study demonstrate that mastoparan stimulated ACTH secretion from permeabilized AtT-20 cells and displayed characteristics consistent with calcium ion- and GTP-y-gamma-S-stimulated ACTH secretion from permeabilized AtT-20 cells. This suggests that in permeabilized AtT-20 cells, mastoparan directly activates GE and that this G-protein may be a heterotrimeric G-protein. This study also suggests mastoparan may be a useful alternative to GTP-gamma-S as a means of directly activating GE.

Involvement of multiple protein kinase C isozymes in the ACTH secretory pathway of AtT-20 cells.

1. The mouse AtT-20/D16-16 anterior pituitary tumour cell line was used as a model system for the study of protein kinase C (PKC)-mediated enhancement of calcium- and guanine nucleotide-evoked adrenocorticotrophin (ACTH) secretion. 2. A profile of the PKC isozymes present in AtT-20 cells was obtained by Western blotting analysis and it was found that AtT-20 cells express the alpha, beta, epsilon and zeta isoforms of PKC. 3. PKC isozymes were activated by the use of substances reported to activate particular isoforms of the enzyme. The effects of these substances were investigated in both intact and electrically-permeabilized cells. Phorbol 12-myristate 13-acetate (PMA, EC50 = 1 +/- 0.05 nM, which activates all isozymes of PKC, except the zeta isozyme), thymeleatoxin (TMX, EC50 = 10 +/- 0.5 nM, which activates the alpha, beta and gamma isozymes) and 12-deoxyphorbol 13-phenylacetate 20-acetate (dPPA, EC50 = 3 +/- 0.5 nM, a beta 1-selective isozyme activator) all stimulated ACTH secretion from intact cells in a concentration-dependent manner. Maximal TMX stimulated ACTH secretion was of a similar degree to that obtained in response to PMA but maximal dPPA-stimulated ACTH secretion was only 60-70% of that obtained in response to PMA or TMX. 4. Calcium stimulated ACTH secretion from electrically-permeabilized cells over the concentration-range of 100 nM to 10 microM. PMA (100 nM), TMX (100 nM) but not dPPA (100 nM) enhanced the amount of ACTH secreted at every concentration of calcium investigated. PMA (100 nM) and TMX (100 nM)significantly enhanced ACTH secretion in the effective absence of calcium (i.e. where the free calcium concentration is nM).5. GTP-gamma-S stimulated ACTH secretion from permeabilized cells in a concentration-dependent manner with a threshold of 1 micro M. PMA (100 nM), TMX (100 nM) but not dPPA (100 nM) increased the amount of ACTH secretion evoked by every concentration of GTP-gamma-S investigated.6. The PKC inhibitor, chelerythrine chloride (10 micro M), blocked the PMA (100 nM)-evoked enhancement of calcium- and GTP-micro-S-stimulated ACTH secretion but did not significantly alter calcium- or GTP-micro-S-evoked secretion itself.7. The present paper indicates that AtT-20 cells express multiple isoforms of PKC and that these act at different sites in the secretory pathway for ACTH secretion. The alpha and epsilon isozymes of PKC can act distal to calcium entry to modulate the ability of increased cytosolic calcium concentrations to stimulate ACTH secretion. This site of action is either at the level of, or at some stage distal to, a GTP-binding protein which mediates the effects of calcium upon ACTH secretion. The beta isozyme of PKC may act ata stage early in the secretory pathway to regulate the cytosolic calcium concentration.

The effects of calyculin A upon calcium-, guanine nucleotides- and phorbol 12-myristate 13-acetate-stimulated ACTH secretion from AtT-20 cells.

1. The mouse AtT-20/D16-16 anterior pituitary tumour cell line was used as a model system for the study of protein phosphatase involvement in the late stages of the secretory pathway for adrenocorticotrophin (ACTH) secretion. The effects of the type 1 and 2 phosphatase inhibitor calyculin A upon calcium-, guanine nucleotide- and phorbol 12-myristate 13-acetate (PMA)-stimulated ACTH secretion from electrically-permeabilized AtT-20 cells were studied. 2. Calyculin A (1 nM-1 microM) inhibited both calcium (10 microM)- and guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) (100 microM)-evoked ACTH secretion from permeabilized cells in a concentration-dependent manner. These effects were maximal with 100 nM calyculin A. 3. ACTH secretion was stimulated from electrically-permeabilized cells when the cytosolic free calcium ion concentration, controlled by calcium-EGTA buffers, was raised over the concentration range of 100 nM to 10 microM. This calcium-stimulated ACTH secretion was inhibited by co-incubation with calyculin A (100 nM). 4. GTP-gamma-S (10 nM-100 microM) stimulated ACTH secretion from permeabilized cells at concentrations greater than 1 microM GTP-gamma-S. Co-incubation with calyculin A (100 nM) inhibited this stimulation of ACTH secretion observed at these concentrations of GTP-gamma-S. 5. PMA (100 nM) significantly stimulated ACTH secretion from permeabilized cells in the absence of either calcium and guanine nucleotides and this action was enhanced by calyculin A (100 nM). Furthermore, an inhibition of GTP-gamma-S (100 microM)-stimulated ACTH secretion observed in the presence of calyculin A (100 nM) was not observed in the presence of PMA (100 nM). 6. The results of the present study indicate that dephosphorylation by phosphatases plays an important role in stimulus-secretion coupling in AtT-20 cells and is involved in mediating the effects of GE upon the secretory apparatus in these cells. Furthermore, the point of regulation of the secretory response by PKC which underlies the ability of PKC to amplify the calcium/GE system may lie distal to both GE and these phosphatases.

Phorbol ester activation of protein kinase C inhibits CNP-stimulated cyclic GMP production in the mouse AtT-20 pituitary tumour cell line.

Preincubation of AtT-20 mouse pituitary tumour cells with the phorbol ester PMA resulted in a concentration-dependent inhibition of CNP-stimulated cyclic GMP production. The phorbol ester analogue 4 alpha phorbol had no inhibitory effect and 24 h preincubations with PMA resulted in a characteristic down-regulation of the response indicating that the inhibitory actions were mediated via the activation of protein kinase C. Forskolin in the presence of the phosphodiesterase inhibitor IBMX stimulated intracellular cyclic AMP concentrations by up to eight fold, but did not alter basal nor CNP-stimulated cyclic GMP production. These results indicate that CNP-stimulated guanylate cyclase activity associated with the GC-B natriuretic peptide receptor expressed in AtT-20 cells is inhibited by protein kinase C.

Effects of protein kinase C activators upon the late stages of the ACTH secretory pathway of AtT-20 cells.

1. The mouse AtT-20/D16-16 anterior pituitary tumour cell line was used as a model system for the study of phorbol 12-myristate 13-acetate (PMA)-mediated enhancement of calcium-evoked adrenocorticotrophin (ACTH) secretion. 2. PMA stimulated ACTH secretion from intact cells in a concentration-dependent manner. Other phorbol esters; phorbol 12,13-dibutyrate (PDBu) and phorbol 12,13-didecanoate (PDD) and diacylglycerol analogues; 1-oleoyl-2-acetyl-sn-glycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DOG) also stimulated ACTH release from intact AtT-20 cells. This would suggest that activation of protein kinase C (PKC) stimulates ACTH secretion from AtT-20 cells. 3. Calcium stimulated ACTH secretion from electrically-permeabilized cells over the concentration-range of 10(-7) M to 10(-5) M. PMA (10(-7) M) enhanced the amount of ACTH secreted at every concentration of calcium investigated. The PKC inhibitor, chelerythrine (10(-5) M) blocked the PMA (10(-7) M)-evoked enhancement of calcium (10(-5) M)-stimulated ACTH secretion but did not alter significantly the calcium (10(-5) M)-evoked secretion itself. This suggests that PKC modulates the secretory response to increases in intracellular calcium but does not mediate the effects of calcium. 4. Guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S, 10(-5) M) stimulated ACTH secretion from permeabilized cells in the absence of calcium and was additive with calcium-evoked ACTH secretion up to a maximum value which could be achieved by calcium acting alone. This suggests that a GTP-binding protein mediates the secretory response to increases in the intracellular calcium. PMA (10-7 M) enhanced ACTH secretion stimulated by the combination of calcium and GTP-gamma-S (10-5 M).5. GTP-gamma-S stimulated ACTH secretion from permeabilized cells in a concentration-dependent manner with a threshold of 10-6 M. PMA (10-7 M) increased the amount of ACTH secretion evoked by every concentration of GTP-gamma-S investigated. Chelerythrine (10-s M) blocked the PMA (10-7 M)-evoked enhancement of GTP-gamma-S (10-4 M)-stimulated ACTH secretion but did not significantly alter GTP-gamma-S(10-4 M)-evoked secretion itself. This suggests that PKC modulates the secretory response to GTP-gamma-S but does not mediate the effects of GTP-gamma-S.6. GTP-gamma-S (10-8-10-4-M) stimulated ACTH secretion from permeabilized cells either in the presence or absence of ATP (5 mM) indicating that its effects on secretion are ATP-independent.7. The results of the present study support the hypothesis that, in AtT-20 cells, PMA is acting at some site distal to calcium entry which modulates the ability of an increase in cytosolic calcium concentration to stimulate ACTH secretion. This site of action is either at the level of or at some stage distal to a GTP-binding protein which mediates the effects of calcium upon secretion.8. PMA, unlike adenosine 3':5'-cyclic monophosphate (cyclic AMP) (Guild, 1991), can stimulate ACTH secretion from permeabilized cells in the absence of added calcium and guanine nucleotides which suggests that PMA and cyclic AMP are acting through distinct mechanisms at this post calcium site of action.

Characterization of natriuretic peptide receptor subtypes in the AtT-20 pituitary tumour cell line.

Receptors for the natriuretic peptide family have been characterized in the adrenocorticotrophic hormone (ACTH)-secreting AtT-20 pituitary tumour cell line. Northern blot analysis detected mRNA transcripts for the guanylate cyclase-linked GC-B receptor subtype. There was no evidence for the expression of either guanylate cyclase-linked GC-A receptor or atrial natriuretic peptide (ANP)-C (clearance) receptor mRNAs. Cyclic GMP production in AtT-20 cells was stimulated up to 200-fold by C-type natriuretic peptide (CNP), which was 10- and 20 times as effective as equivalent concentrations of brain natriuretic peptide and ANP respectively. Cyclic GMP dose-response curves to CNP failed to show any signs of saturation even at concentrations up to 30 microM, indicating a relatively low affinity of CNP for the GC-B receptor. Although CNP induced large stimulations in cyclic GMP production, specific binding of [125I-Tyr0]CNP could not be demonstrated in AtT-20 cells. The absence of specific binding with this radiolabelled analogue is possibly due to a reduced affinity for the GC-B receptor, as CNP analogues with N-terminal modifications such as [Tyr0]CNP and [127I-Tyr0]CNP exhibited reduced abilities to stimulate cyclic GMP production in these cells. Despite elevating cyclic GMP levels, CNP had no effect on basal or corticotrophin-releasing factor-stimulating ACTH release from the cells. These results show that the guanylate cyclase-coupled GC-B receptor is the only natriuretic peptide receptor subtype expressed in AtT-20 cells. Although CNP can markedly stimulate cyclic GMP production in these cells, there is incomplete expression of the normal natriuretic peptide-induced inhibitory pathway of ACTH secretion at some point distal to the production of cyclic GMP.

Atrial natriuretic peptide effects in AtT-20 pituitary tumour cells.

Whether atrial natriuretic peptide (ANP)-evoked inhibition of corticotrophin-releasing factor (CRF)-stimulated ACTH secretion was also manifest in ACTH secreting AtT-20 pituitary tumour cells was investigated. ANP stimulated increases in cGMP accumulation at concentrations of the peptide above 10(-8) M which indicates the presence of the ANP receptors on these cells. CRF stimulated a concentration-dependent increase in ACTH secretion from AtT-20 cells which was unaffected by ANP, 8-bromo-cGMP, or sodium nitroprusside (SNP). Calcium stimulated a concentration-dependent increase in ACTH secretion from electrically permeabilised cells which was unaffected by co-incubation with cGMP but potentiated by cAMP. These results reveal the presence of ANP receptors on AtT-20 cells but suggest that an incomplete expression of the stimulus-secretion coupling mechanisms for ANP, at some point after cGMP production, prevents the effects of natriuretic peptides upon ACTH secretion being manifest in these cells.

The interaction between noradrenaline and ATP upon polyphosphoinositide metabolism and contraction in tail arteries from normo- and hypertensive rats.

The effects of, and interaction between, noradrenaline and alpha,beta-methylene ATP upon polyphosphoinositide (PPI) breakdown, investigated by measuring the accumulation of inositol phosphates, and contraction, were studied in tail arteries from normo- (WKY) and spontaneously-hypertensive (SHR) rats. Noradrenaline (10(-7)-10(-3) M) evoked a prazosin (10(-6) M)-sensitive, concentration-dependent increase in total inositol phosphate accumulation in both WKY and SHR rats. No significant differences were observed in either the maximal response or in the concentration range over which noradrenaline evoked this response, between these two populations. Noradrenaline (5 x 10(-7)-5 x 10(-5) M) evoked a concentration-dependent contraction of arteries from both SHR and WKY rats. The responses to noradrenaline were about 2-fold greater at all effective concentrations of noradrenaline in SHR compared with WKY rats. alpha,beta-Methylene ATP (10(-6) M) did not alter noradrenaline-stimulated total inositol phosphate accumulation, in arteries from either SHR or WKY rats, measured either as the maximal response or as the EC50. alpha,beta-Methylene ATP (5 x 10(-6) M), by itself, evoked a contractile response, which was quantitatively similar in SHR and WKY rats, and was additive with the contractile responses to noradrenaline (5 x 10(-7)-5 x 10(-5) M). The maximum response produced by a combination of noradrenaline and alpha,beta-Methylene ATP was quantitatively similar to that produced by noradrenaline alone. No evidence of synergism between alpha,beta-Methylene ATP and noradrenaline upon contraction was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of noradrenaline and adenosine 5'-triphosphate on polyphosphoinositide and phosphatidylcholine hydrolysis in arterial smooth muscle.

1. The effects of noradrenaline and alpha,beta,methylene adenosine 5'-triphosphate (alpha,beta,methylene ATP) on polyphosphoinositide metabolism, phosphatidylcholine hydrolysis and contraction in rabbit saphenous arteries were investigated. The effect of noradrenaline upon polyphosphoinositide metabolism was also investigated in the rat tail artery. 2. Noradrenaline (10(-7)-10(-4) M) evoked a concentration-dependent increase in total inositol phosphate accumulation in the rat tail but not in the rabbit saphenous artery. Propranolol (3 x 10(-6) M) did not alter this result in the rabbit saphenous artery. In addition, alpha,beta,methylene ATP (10(-6) M) significantly increased total inositol phosphate accumulation in the rabbit saphenous artery, while potassium chloride (8 x 10(-2) M) was ineffective. 3. Phorbol 1,2-myristate 1,3-acetate (3 x 10(-8) M) enhanced noradrenaline (10(-2)-10(-4) M)-evoked contractions in rabbit saphenous artery. The contractile responses to potassium chloride (1- 16 x 10(-2) M) in tissues treated with 6-hydroxydopamine (5 x 10(-4) M), in vitro, were unaffected by these concentrations of the phorbol ester. 4. Noradrenaline (10(-6)-10(-4) M) evoked a concentration-dependent increase in the levels of choline and choline phosphate, but not in those of glycerophosphocholine, in the rabbit saphenous artery. Choline levels increased significantly over the first 15-30 s then declined to control levels within 2 min of addition of noradrenaline (10(-5) M). A smaller initial rise in choline phosphate levels (15-30 s) was followed by a larger secondary rise at 2-4 min.5. alpha, beta, methylene ATP (10-1_ 0-4 M) also evoked a concentration-dependent increase in the levels of both choline and choline phosphate, but not those of glycerophosphocholine, in the rabbit saphenous artery. alpha, beta, methylene ATP (10-4 M) significantly increased levels of both of these products within the first 15-30 s of addition of the drug; these levels reached a stable plateau 1 min after addition.6. The maximum accumulation of choline or choline phosphate evoked by either noradrenaline or alpha, beta, methylene ATP, acting alone or in combination, was not significantly different. No evidence of synergism between noradrenaline and alpha, beta, methylene ATP was observed.7. This study demonstrates that each of the co-transmitters in the rabbit saphenous artery, noradrenaline and adenosine 5'-triphosphate (ATP), promote phosphatidylcholine hydrolysis. Noradrenaline seems to rely on phosphatidylcholine hydrolysis to mediate its contractile effects, whilst ATP promotes both polyphosphoinositide and phosphatidylcholine metabolism suggesting that multiple signal-transduction mechanisms are involved in stimulus-contraction coupling in this artery.

Mechanical and biochemical effects of individual co-transmitters in rat tail arteries.

1. The effects of (1) nerve stimulation (NS, 0.5 ms, 1-16 Hz, supramaximal voltage, 20 s) upon vasoconstriction, as measured by changes in perfusion pressure and (2) exogenously added co-transmitters noradrenaline (NA) and alpha, beta,-methylene ATP (alpha, beta, MeATP) upon polyphosphoinositide (PPI) breakdown, and vasoconstriction, were studied in rat tail perfused arteries or arterial rings. The interaction between NA and ATP upon contraction of artery rings and inositol phosphate (IP) accumulation was also studied. 2. Nerve stimulation evoked vasoconstriction in rat tail arteries. These pressor responses were largely (approximately 80%) blocked by the alpha 1-adrenoceptor antagonist, prazosin (10(-6) M), but not significantly affected by desensitization of P2x-purinoceptors by prior, repeated (five times) addition of alpha, beta, MeATP (10(-6) M). 3. Noradrenaline evoked a prazosin (10(-6) M)-sensitive, concentration-dependent, increase in both perfusion pressure and total inositol phosphate (IP) accumulation over the same concentration range (10(-6)-10(-4) M). The amplitude of the pressor responses to NA were about 80% of those obtained to nerve stimulation (0.5 ms, 1-16HZ 20s supramaximal voltage). 4. alpha, beta,-methylene ATP (10(-7)-10(-5) M) evoked a concentration-dependent increase in perfusion pressure which, at maximum, was equivalent in amplitude to approximately 20% of that obtained to nerve stimulation. Concentrations of the nucleotide greater than 10(-5) M were required to stimulate total IP accumulation. 5. The effects of NA (10(-6) M) and alpha, beta, MeATP (10(-5) M) upon contraction of artery rings and IP accumulation were additive and no evidence of synergism between them, on either parameter, was obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Noradrenaline-stimulated inositol phosphate accumulation in arteries from spontaneously-hypertensive rats.

1. The effects of noradrenaline upon polyphosphoinositide (PPI) breakdown was investigated by measuring the accumulation of inositol phosphates (IPs) in tail arteries from normo- (WKY) and spontaneously-hypertensive (SHR) rats. 2. Noradrenaline (10(-7)-10(-3) M) evoked a concentration-dependent increase in total IP accumulation in both WKY and SHR rats but no significant differences between the populations were detected. 3. In contrast, significant differences in the accumulation of the individual IPs, which contributed to the total IP, occurred. A significantly greater noradrenaline-stimulated accumulation of inositol trisphosphate (IP3) was observed in tissues from SHR compared with those from WKY rats at each effective concentration of noradrenaline. This was paralleled by an equivalent reduction in inositol monophosphate (IP1) accumulation, consistent with the lack of a significant difference in noradrenaline-stimulated total IP accumulation between the two populations. 4. In time course studies, an enhanced noradrenaline-induced accumulation of IP3, in SHR compared to WKY rats, occurred from the earliest time point studied after the addition of the catecholamine both in the presence and absence of LiCL (10 mM). In the presence of LiCl (10 mM) no significant difference in noradrenaline-evoked total IP accumulation between SHR and WKY rats was observed; in the absence of LiCl noradrenaline-evoked a greater total IP accumulation in SHR than in WKY rats at all time points investigated. 5. These studies suggest that the main reason for the enhanced noradrenaline-induced accumulation of IP3 in arteries from SHR rats is a reduced rate of dephosphorylation of both IP3 and inositol bisphosphate (IP2) rather than a greater formation of IP3 from PPIs.6. This enhanced accumulation of IP3 may result in an increased calcium mobilisation accounting for the increased contractility to noradrenaline of tail arteries from SHR as compared with those from WKY rats.

Differential effects of suramin on the coupling of receptors to individual species of pertussis-toxin-sensitive guanine-nucleotide-binding proteins.

The anti-helminthic drug suramin inhibited the basal high-affinity GTPase activity of both C6 BU1 glioma and NG 108-15 neuroblastoma x glioma hybrid-cell membranes with an IC50 (concentration causing half-maximal inhibition) value close to 30 micrograms/ml. This effect was shown to occur via a non-competitive mechanism in which the binding affinity of the G-proteins for GTP was not altered, but the maximal velocity of the subsequent hydrolysis was reduced. In NG 108-15 membranes, both opioid peptides and foetal-calf serum stimulated high-affinity GTPase activity in a pertussis-toxin-sensitive manner. These effects have previously been shown to be mediated by different G-proteins [McKenzie, Kelly, Unson, Spiegel & Milligan (1988) Biochem. J. 249, 653-659]. Suramin completely prevented the opioid-peptide-stimulated increase in GTP hydrolysis, but did not prevent the opioid peptide from binding to its receptor. Suramin, however, did not block the foetal-calf-serum-stimulated GTPase response. This selective action of suramin provides further evidence for distinct roles for two separate pertussis-toxin-sensitive G-proteins in signal transduction in NG 108-15 membranes and provides the first evidence for a selective effect of a drug on the functions of different G-proteins.