476

BIOCHEMICAL SOCIETY TRANSACTIONS

Skeletal muscle

L

'non-hepatocyte' cell populations and that such cells d o not show corresponding decreases in mRNA for G, 2 and G,. Interestingly, in adipocytes we observed a substantial increase in the number o f transcripts for the a-subunits of G, 1 and of G, 3 ( - 4-fold), but n o change in either GI 2 o r G, . mRNAs for the a-subunits of G, 3 and G, were decreased by 50% and 40%, respectively, in skeletal muscle from diabetic animals, with no change in G, 2. G, 1 mRNA was not detected. We suggest that diabetes causes alterations in the levels of mRNA for the a-subunits of certain G-proteins, in a tissuespecific manner and that this may be relevant to observed alterations in signal transduction mechanisms in the affected tissues.

-

Adipocytes

0

100

200 300 400 Transcripts in diabetic tissues (relative to control as I 00"/0) Fig. I . Ej'ect of' streptozotocin-induced diabetes on the levels of G-protein a-subitnit mRNAs

Northern blots of total RNA, isolated from different tissues from control (untreated) and diabetic rats, were probed with radiolabelled a-subunit-specific oligodeoxynucleotides. Analysis of hybridization of the probes to specific mRNAs was by scanning densitometry of autoradiographs. Values for RNA from diabetic tissues have been expressed as a percentage of control values. mRNA for the a-subunit of GI 2 ( - 25"/0) was observed, there was no reduction in mRNA for G, a-subunit and transcripts for the a-subunit of G, 1 were detected, but at very low levels. We suggest that GI 1 in liver is derived from the

I . Gilman. A. G. ( I 9x7) Annu. H e i : H i d w m . 56. 6 15-64') 2. Gawler. D., Milligan. G.. Spiegel, A. M..Unwn. C. G . & Houslay. M. D. (1987) Nutitre (London) 327,229-232 3. Lochrie, M. A. & Simon, M. 1. ( I9 X X ) Hiochc,mi.stn 27, 4957-4965 4. Codina, J., Stengel. D., Woo. S. L. C. & Birnbaumer, L. ( I O X 6 ) FEBS. Lett. 207. I X7- I92 5. Fong. H. K. W., Hurley, J . B.. Hopkins, R. S., Miake-Lye. R., Johnson, M. S., Doolittle, R. E & Simon, M. 1. ( 1 986) /'roc. Nutl. Acnd. Sci. U.S.A.83,2162-2166 6. Fong, H. K. W., Amatruda. T. T., Birren. B. W. & Simon. M . 1. (1987) I'roc. Nutl. A c d . Sci. U.S.A.84, 3792-3796 7. Hurley, J. B., Fong, H. K., Teplow, D. B., Dreyer, W. J. & Simon, M. 1. (1984) I'roc. Nutl. Acud. Sci. U.S.A.81,6948-6952 8. Robishaw. J. D.. Kalman. V. K.. Moomaw, C. R. & Slaughter. C. A. ( 1989)J. B i d . ('hem. 264, I S 758- I S 76 I 9. Gautam, N., Baetscher, M., Aebersold, R. & Simon, M. 1. ( 19x9) Science 244.97 1-974

Received 20 November 1989

Effects of capsaicin on glucose metabolism in isolated incubated skeletal muscle in vitro BRENDAN LEIGHTON and ELIZABETH FOOT Depurtment of'Biochemistry, University of Oxford, Oxford 0x1 3QU, U.K. The neuropeptide calcitonin gene-related peptide (CGRP) is a 37 amino acid peptide which is localized in both sensory nerves [ I ] and in the motor end-plate axon terminal [2] in skeletal muscle. CGRP has about 50% primary amino acid sequence identity with the novel pancreatic hormone amylin [3]. Both peptides are potent inhibitors of both basal and insulin-stimulated rates of glycogen synthesis in incubated skeletal muscle preparations [4-71. Indeed, it is hypothesized that an abnormality in amylin/CGRP homoeostasis may underlie pathological insulin resistance found in many disease states, such as non-insulin-dependent diabetes mellitus [4]. Electrophysiological studies have demonstrated that low levels (1-10 ,UM) of capsaicin (the pungent ingredient in capsicum) exert potent excitor effects on sensory neurones (i.e. non-myelinated afferent nerve endings of the C-fibre type), Furthermore, capsaicin affects a variety o f processes (e.g. contractility of beating atrium in vitro), but there is compelling evidence that these effects are produced via CGRP [8-10]. Thus, if skeletal muscle is treated with capsaicin, to Abbreviations used: CGRP. calcitonin gene-related peptide; EDL, extensor digitorum longus.

release CGRP from sensory nerves, this may cause inhibition of insulin-stimulated glycogen synthesis. Consequently, soleus and extensor digitorum longus (EDL)muscle preparations were incubated with capsaicin (10 ,UM) and the insulinstimulated rates of lactate formation and glycogen synthesis were measured. Soleus muscles were prepared and incubated [ 111 from Wistar rats (140 g). Muscles were incubated under tension (on stainless-steel clips) in Krebs-Ringer bicarbonate buffer containing 5.5 mM-glucose (plus 0 . 3 pCi of [U-14C]glucose/ ml), 1% (w/v) defatted bovine serum albumin and a range of concentrations of insulin (see Table 1 ). After incubation for 60 min, muscles were removed and rapidly frozen in liquid N,. The concentration of lactate in the incubation medium [ 121 and the amount of [U-'JC]glucose incorporated into glycogen [ 131 were measured. Insulin stimulates two major processes in isolated incubated muscle preparations, namely glycogen synthesis and glucose transport and subsequent conversion to lactate [5,6, 141. The concentration of insulin which normally yields a half-maximal response for both processes is 100 punits/ ml. The effects of capsaicin ( 1 0 ,UM)on insulin-stimulated rates of lactate formation and glycogen synthesis in incubated soleus or EDL muscle preparations are given in Table 1. Capsaicin significantly inhibited the rate of glycogen synthesis in both muscle preparations. However, capsaicin stimulated the rate of lactate formation only in the EDL muscle in vitro. Qualitatively similar results have been 1990

477

633rd MEETING. LONDON Table 1 . Efecxs of insulin (IM)p-unitsltnl) or1 the rates of' Icic~tcitefbrtncitiori citicl glwogeti ,sytithe.si.s in i.solatec1 stripped soleus cind intact (EI)L) tnuscle iticdxitccl in /lie trhsetic~eor presence of cupsaiciti ( 1 0 ~ ~ )

All values represent means k S.E.M. for at least eight separate experiments. Statisticnlly significant differences (Student's t-test) between incubations in the absence or presence o f capsaicin are denoted by *I>< 0.05. Kate of glycogen synthesis ( p m o l 'glucosyl' unita/h pel- gi

Rate of lactate formation (pnol/h per g wet wt)

Control

Capsaicin

Soleus

EDL

Soleus

I 7 . I 2 f0.42 12.72 f 0 . 5 0

9.73 0.7 I 12.27 0.7 I *

*+

3.45 0 .I I 2.03 + 0 3 P

obtained with C G R P in the incubated stripped soleus and intact E D L preparations it1 vitro [4-61. This raises the possibility that C G R P may be an important physiological regulator o f glycogen metabolism in skeletal muscle. I.

2, 3. 4.

5. 6.

+

EI>I. 2. I6 f 0 .I Y I .62 f 0 .I5*

7. Cooper, G. J. S.. Leighton. H.. I>imitriadis. G. I>., Parry-Billings. M., Kowalchuk, J . M.. Howland. K.. Kothhnrd. J . 13.. Willis. A. C. & Keid. K. H. M. ( 1988) /'roc.. Nrirl. Ac.tic/. .Sc,i. CI..S.A. 85. 7 76 3 - 7 7 6 0

8. Maggi, C. A,, Snnticioli, P., ~I'heodorsson-Norheim,E. NC Mcli. A. ( 1987) Ncirrosc.i. I.(,//.78. 683-688 9. Miyauchi, -1.. Ishikawa. T., Sugishitn, Y., Saito. A. NC Goto. K. Ohlcn. A,. Lindholm. L., Staines. W.. Hokfelt. I...Cuello. A. C.. I'htirrti~ic.o/.10. 075-088 Fisher. J. A. bi Hedqvist. P. ( 1987) N t i i i r i ~ r i - , S ~ , l i ~ e i ~ ~ r ~ i , r ~( 1. sYX7) C'cirt/ior,ti.sc~. 10. Franco-Cereceda, A. NC Lunhcrg. J . M. ( I Y 8 5 ) N(iioi.iwArch. /'hcirmacd. 336. 87-93 .S~~lrr,iiec/c~hc~r~,s Arc,li. l'litirr?rti(~o/. 3 3 1. 146- I 5 I Tithami. K. Y.. Kawai. Y.. Uchida. S.. Tohyama. M.. Shiotani. Y.. I I . Esninal. J.. Dohm. C;. L. NC Ncwsholmc. E. A . ( I YX3 I H i o c . l i c w i . Yoshida. H., Emson. P. C., Girgis, S.. Hillyard, C. J. & MacJ. 212.453-458 Intyrc. I. C. ( 1985) Neiirosci. l . i p / / . 60. 227-230 12. kneel. P. C'. & Jane\. J . H. j I Y78) / t r i t i / , H i o c hcnr. 88. 475-484 Cooper. G . J. S.. Willis. A. C.. Clark. A,. Turner. K.. Sim. K. B. 13. Cuendet. G.. Loten. E.. Jcanrenaud. 13. CQ Kenold. A. ( 1070) .J. NC Reid. K. H. M. ( 1987) /'roc.. M i / / . /tuit/. S c i . U.S.A. 84. C ' / i r i , / ~ i i ~ . s5t8. . 1078- I088 8628-8632 14. Challiw. K. A. J.. Leighton. 13.. Lozcman. F. J. NC Newsholme. Lcighton. H. bi Cooper. G. J. S. ( I Y88 I Ntitiire ( l m i c l o r i ) 335. E. A. ( I 987) in 7k)pics rod I'c,r.\pi~c.ti~u~.\ iti /tdiwo.\itw Hiwrircli h 32 -635 (Gcrlnch. E. & l3cckcr. 13, I-.. eds.). pp. 775-285. SpringerLxighton. H.. Dimitriadis. G. I>.. Parry-Billings. M.. Lozeman. Verlag, Berlin, Heidelberg F. J . bi Newsholme. E. A. ( I Y89) H i o c h c m . J. 261, 383-387 Lcighton. H.. Foot. E. A,, Cooper. G . J. S. di King, J . M. ( 19x9) Received 24 November I080 k'l.'tj.S l.cl/t. 249. 357-4 I3

Inhibition of platelet membrane hormone-transducing GTPases by cyclic GMP-dependent protein kinase A N D R E W C . NEWBY, ROBERT 0.M O R G A N and LYNDA M. BLAYNEY lkpcirtimvit of C iircliolo~?, Utiitwsity of' Wciles College of' Mcdic~irie.Iiearli I'cirk, C'cird$fC'F-/ 4 X N . U.K . Endothelium-derived nitric oxide and nitric oxide-liberating drugs (e.g. nitroprusside. molsidomine) inhibit platelet aggregation. shape change and adhesion t o endothelium o r subendothelium [ 1-41. Nitric oxide thereby contributes to endothelial athrombogenicity. while nitrovasodilator drugs may have hitherto little exploited antithrombotic properties. Nitric oxide activates platelet-soluble guanylate cyclase which increases cyclic G M P concentration and in turn activates the cyclic GMP-dependent protein kinase (kinase G). This enzyme by phosphorylating largely unidentified proteins inhibits agonist-induced calcium influx and, at higher concentrations, calcium mobilization. which probably accounts for inhibition o f platelet activation [2,4].To seek to explain the dual inhibition o f calcium influx and mobilization, we examined the hypothesis that kinase G inhibits the signal-transducing GTPases involved in activating these processes. We tested the effect of purified kinase G on the agonist-stimulated GTPases o f platelet membranes [ 5,6]. Human platelets were obtained free of plasma [4] and membranes were prepared and resuspended t o 1.5 mg of protein/ml [ 5 1. Aliquots o f suspension were stored at - 70°C until needed. GTPase activity was measured at 30°C [ S ] . Pig Vol. 18

lung kinasc G (specific activity 1.3-5.1 m-units/mg o f protein) was prepared 171 and activity was determined at 30°C as previously described 1x1 with 1.5 mg o f histone IIA/ ml (Sigma Chemical Co.. Poolc. Dorset) as substrate. O n e unit of enzyme transferred I pmol o f P,/min. Kinase-G o r buffer 171 was preincubatcd at 30°C with membranes (0.15 mg of protein/ml) with o r without 1 pM-cyclic G M P in GTPase assay medium (containing ATP; IS]) for 10 min before adding G T P for 0 (blank) o r 5 min (experimental). Control incubations were also conducted without membranes which revealed contaminating GTPase activity in one of four preparations o f kinase G. Unstimulated platelet GTPase activity was 24 k 3 pmol/ min per mg o f protein (mean ~ s . E . M . ,t i = 1 I ; cf. 1.5. 61).T h e activation obtained with various agents (expressed as a percentage of unstimulated activity) was for: 2 units of thrombin/ml, 43 k 1 0 % ( t i = 4); 2 0 units o f thrombin/ml, 5 7 f 6% ( t z = S ) ; 20 ng of platelet activating factor/ml, 6+4"/0 ( t z = 3); 2 0 0 ng o f platelet activating factor/ml, 29 f 2% ( I I = 3); 0.0 1 mwprostaglandin E , , 18 k 3% ( t i = 4); 0.1 mMprostaglandin E , , 4 1 f 7% ( t i = 4); 0.0 1 mwadrenaline1 k 8% ( 11 = 3) and 0. I mwadrenaline 17 k 5% (ti = 4).T h e potency and efficacy o f these agents was similar to values previously reported [ 5,6]. Preincubating membranes with 170 p-units of kinase G/ml resulted in approximately 20% inhibition of unstimulated GTPasc activity (Fig. 1 ) and inhibition was not signifi-