- Email: [email protected]
Endogenous digitalis-like compounds: putative regulators of the sodium pump
TINS - September
I984
oocyte, but these chains are assembled into complete AChRs that are inserted into the surface membrane where they function normally. Cells maintained in culture- for example. the neuroblastoma cell line Nl8 which lacks AChRs - can be used for microinjection experiments instead of oocytesU.‘-‘. For many experiments oocytes are preferable because they are so large (almost 1mm in diameter) and have such a large number of ribosomes: injection of a single oocyte can produce large enough quantities of protein to be easily detected by antibodies and used for some biochemical experiments. Cultured cells, however. are superior for some cell biological experiments (e.g. how does altered protein structure affect assembly and insertion of a channel) and possibly for physiological experiments (e.g. single channel record ing). For example. oocytes might in some circumstances yield too low a surface density to be useful for single channel recording but one could study, in principle at leasf just one AChR copy in a cultured neuron by using the whole cell recording configuration (see Ref. 14). In summary. then, the relationship between AChR structure and function can be investigated by altering the genetic blueprint for the protein, having this blueprint expressed in cells, and studying the functional implications of alterations in the modified proteins. This method was described here in the context of a specific
307 example, but clearly the approach is a general one. Other channel types. recep tors and enzymes involved in second messenger systems (e.g cyclic nucleotides). cell surface proteins that participate in cell-cell recognition, mechanisms of pro tein sorting and insertion into the surface membrane, and proteins responsible for exocytotic events and membrane recovery can all, in principle. be studied in a precisely analogous way. Although the method is a powerful one, a number of complications are evident. First, the DNA specifying a protein to be studied must have been cloned and sequenced; so far, the only channel protein for which this is true is the AChR Next. the manipulations involved can be rather time consuming with several weeks of work needed for making a single modification. Finally, the interpretation of the results is not at all automatic. As with other methods. several different tests of each hypothesis. by making various mod5 cations in the protein structure, will be required and predictions about functional implications of the changes made will have to be made with care and imagination. In conjunction with other approaches. though. site directed mutagenesis should provide a key for unravelling the molecular basis for nervous system function. Reading list 1 Kao. P.N.. Silver. M.L.
Dwork. A.J.. Kaldany. R-RJ.. Wideman J.. Stein S. and Karlin.
10
II I2 13 14
A (1984) J BiaL Chem 259. (in press) Walker. J. W.. Lukas, R J. and McNamee. M. G. ( I 98 I ) BiochemisfqJ 20. 2 19 I-2 I99 Dionne. V. E.. Steinback J. H. and Stevens. C. F. ( 1978)J. PhysioL {London) 28 I. 42 1444 Noda. M., Takahashi. H, Tanabe. T.. Toyosato. M. Furutani. Y.. Hirose. T., Asai. M.. Inayama. S.. Miyata. T. and Numa S. (I 982) Nature (London) 299, 793-797 Winter. G., Fersht A R. Wilkinson A. J.. Zoller. M. and Smith. M. ( 1982) Nature (Low don) 299.156-158 Green M. R. Mania& T. and Melton. D. A. (1983) Cell 32,681-694 Mom& S. M.. Pettersson. I.. Hinterberger. M.. Karmas, A. and Steitl J. A (1983) Cell 33. 509-5 18 Sumikawa. K.. Houghton. M.. Emtage. J. S.. Richards, B. M. and Barnard. E. A. (1981) Nature (London) 292. 862-864 Barnard. E. A. Miledi. R and Sumikawa K (1982) Proc R Sot. London B215.241-246 Mishina. M., Kurosaki. T.. Tobimatsu. T.. Morimoto. Y.. Noda. M.. Yamamoto. T.. Terao. M.. Lindstrom. J.. Takahashi. T.. Kuno. M. and Numa. S. ( 1984) Nature (London) 307.604608 Gurdon. J. B. and Melton. D. A ( I98 I ) Annu Res Gener 15. 189-218 Graessmann A.. Graessman. M. and Mueller. C. ( 1980) Methods Enz.vmoL 65. 8 16-825 Stacey. D. W. and Allfrey. V. G. (1916) Ce119. 725-732 Sakmann B. and Neher, E. ( 1983) in Single Channel Recording, Plenum Press. New York CHARLES
F. STEVENS
Section ofMolecularNeurobiolog.x Yale Universir?, School of Medicine. 333 Cedar SrreeC New Haven. CT 06510. USA.
induce positive inotropic effects in hearts or inhibit the short circuit current in amphibian epit.helium’O. Many investigators have assumed that an extract is digitalis-like if it produces one or more of these effects. However, many substances The quest for endogenous digitalislike factors has been given new impetus by the that are not inhibitors of the Na’ pump fmding that theplasma activity of theseputativefactors mayparticipate in the renal may appear to have‘digitalis-like’ propercontrol ofjluid and electrolyte metabolism and may play a role in the etiology of ties, as judged from their ability to produce hypertension The brain may be intimatelv involved in controlling theactivity ofthese cardiotonic effects or inhibit trans-epithe lial sodium transport Conversely, some factors. inhibitors of the Na’ pump, such as Ca” The discovery that enkephalins are endo exchange mechanism’. or vanadate, would not be considered genous opiates has accelerated the search There has been much speculation about ‘digitalis-like’. for other endogenous factors in mammalian the existence of endogenous factors with Efforts to isolate substances with DLA systems where specific receptors with ‘digitalislike’ activity (DLA) since Schatz- have generally begun with large quantities high affinity for exogenous substances, mamY first described the inhibition of of source material from humans or animals have been identified A particularly good active monovalent cation transport in red (plasma or urine, or homogenates of cells by digitalis glycosides. Examples of various tissues, e.g brain kidneys, adrenals, example is the interaction of the digitalis glycosides and other cardiotonic steroids ‘digitalis-like’ properties include: (a) in- or amphibian skin), often under different with the sodium pump. The digitalis glyco hibition of ouabainsensitive cellular ca- physiological conditions. In most cases, sides have been used for two centuries to tion fluxes3.? (b) inhibition of( Na’ i- K’) extracts have been chromatographed and treat congestive heart failure. Their -ATPase activity in isolated plasma mem- concentrated to increase DLA, which is mechanism of action is uncertain, but is brane preparation9.6.7.8; (c) competition usually quite low in the starting materials. believed to involve the binding to, and between radiolabelled digitalis glycosides However, these procedures may also amp subsequent inhibition of, the cardiac sar- and putative ‘digitalis-like’ factors, for lify the activities of interfering substances, either membrane-associated binding colemmal sodium pump; this indirectly often in a non-linear manner, so that they increases cardiac contractility by promotsites6.8.9or antibodies raised against specific are then mistaken for the substance(s) of digitalis glycosides6; and (d) ability to interest Furthermore, both the bioassays ing calcium accumulation via a Na’/CaZ’
Endogenous digitalis-like compounds: putative regulators of the sodium pump
0
19H4. Elrcwc :r Saencc
Pubhcatums
B.V.. Amsterdam
0.178 - 5Y I2/84/S02.00
TINS - September 1984
308 and biochemical methods that are fre quently used to detect DLA are, themselves, subject to many types of interference. These may be some of the reasons why no endogenous digitalislike sub stances have been isolated and characterized in the 30 years since the discovery of the interaction between the digitalis glyco sides and the Na’ pump, and the 15 years since the first report of an endogenous inhibitor of (Na’ + K’)-ATPase. The purification of digitalislike substances will obviously depend upon the establish ment of reliable assays, that can provide predictable results when several of the criteria mentioned above are tested. Since the pioneering observations of de Wardener and Mills”, numerous investigators have convincingly demonstrated that extracellular fluid (ECF) volume expansion triggers a natriuresis that can not be explained by modulation of aldo sterone secretion, or alteration of glomeru lar filtration rate. Extracts of plasma or urine from volume-expanded animals and man were found to contain one or more factors with DLA, as identified by the aforementioned criteria For example, a natriuretic factor was found in the urine of salt-retaining (volume-expanded) uremic patients, but not in that of salt-losing nephrotics I2. Furthermore, there is evidence that plasma from uremic patients contains elevated levels of a circulating inhibitor of Na’ pumpsL3J4. This substance should have a natriuretic effect analogous to that produced by digitalis glycosides, that can be explained by inhibition of renal tubule Na+ pumps - especially in the distal tubule’4, where the final composition of the urine is determined. Dahl15 was the first to suggest that a circulating natriuretic factor might be involved in the etiology of hypertension. Later workers postulated that this natriuretic, hypertensinogenic hormone might be a Na+ pump inhibitor, and that it might play a central role in the genesis of some ‘volumedependent’ forms of experimental hypertensionI and of essential hypertension in man”~ 18.Recent direct evidence shows that patients with essential hypertension have elevated plasma levels of a circulating Na’ pump inhibito?.‘. This substance could promote the rise in blood pressure by providing increased amounts of Ca*+, as well as Na+, to vascular smooth muscle and sympathetic neurones via a Na+/Caz+ exchange mechanism17~‘g. Numerous animal experiments indicate that the brain has a key role in controlling the activity of this natriuretic agent In rats, lesions in the anteroventral region of the hypothalamus adjacent to the third ventricle (AV3V), abolish the secretion
of a Na” transport inhibitor that usually is present in the plasma in mineralocorticoid (plus salt)-induced hypertension4, and foL lowing ECF volume expansionlO. These AV3V lesions also lead to hypematremia, and they attenuate or alleviate the rnineralo corticoid-induced and other types of saltdependent hypertension The rare human syndrome, ‘essential hypematremia’, that is usually associated with damage to the hypothalamic region of the brainzO, may be a consequence of impaired secretion of a digitalis-like material with natriuretic and hypertensinogenic properties19. Further evidence that the regulation of salt and water balance is far more complex than previously suspected comes from the discovery of several (cardiac) atria1 pep tides with natriuretic activity2 ‘. However, these substances cause vasodilation**, and do not inhibit (Na’ + K+)-ATPase23, therefore they are not digitalis-like. The convergence of these multiple lines of evidence, along with the newly-recognized pathophysiological implications, have helped to generate excitement about the role of digitalis-like substances in cation transport, and salt and water metabolism The key to unravelling these fundamental mechanisms is the isolation and characterization of the endogenous digitalislike compounds. Reading list I Baker. P. F., Blaustein M. P.. Hodgkin A L and Steinhardt. R A ( 1969)J Ph.vsioL (London) 200.497-527 2 Schatzmann. H. J. (1953) Helv. PhysioL Pharmacol Acru 11. 346-354 3 Poston, L., Sewell R B.. Wilkinson S. P.. Richardson, P. J., Williams, R. Clarkson, E. M, MacGregor. E. A and dewardener, H. E. (1982)Br. Mea! J. 282.847-849 4 SonguMize, E., Bealer. S. L and Caldwell. R W. (1982) HJpertension 4,575-580 5 Gonick. H. C., Kramer, H. J., Paul, W. and Lu, E. (1977) Clin Sci MoL Med 53, 329-334 6 Gruber, K. A. Whitaker, J. M. and Buckalew, V. M.. Jun (1980) Nature(London) 237,143145
-
7 Hamlyn J. M, Ringel. R. Schaeffer. J., Levinson, P. D.. Hamilton B. P.. Kowarski. A A and Blaustein, M. P. (1982) Nature. London 300.650-652 8 Shimoni. Y., Gotsman. M.. Deutsch, J.. Kachalsky. S. and Lichtstein D. ( 1984) Nature (London) 307. 369-371 9 Devynck M. A., Pemollet. M G., Rosenfeld, J. B. and Meyer, P. (1983) BI: Med J. 287. 631-634 IO Bealer, S. L. Haywood J. R. Gruber, K. A. Buckalew, V. M. Jun., Fink, G. D.. Brody. M. J. and Johnson. A. K (1983) Am .L PhysioL 244. R5l-R57 I I de Wardener, I-I E., Mills, I. H.. Clapham. W. F. and Hayter. C. J. ( I96 I ) C/in. Sci 2 I. 249-258 12 Bourgoignie. J. J., Hwang, K H., Ipakchi, E. and Bricker, N. S. (1974) J. Clin Invest 53. 1559-1567 I3 Smith, E. K M. and Welt. L G. (1970) Arch Intern Mea! 126.827-830 I4 Fine, L G.. Bourgoignie, J. J.. Hwang K. H. and Bricker, N. S. (1976) J. Ciin Invest 58. 590-597 I5 Dahl, L K.. Knudsen, D. K D. and lwai J. (1969) Circ. Reseurch (Suppl r). I-21 - I-33. 24-25 I6 Haddy, F. J. and Overbeck. H. W. ( 1976) Life Sci 19.935-948 17 Blaustein, M. P. ( 1977) in Excitation-Contmction Coupling in Smooth Muscle (Casteels, R, Godfraind T. and Ruegg, J. C., eds), pp. IOI108. Elsevier/North Holland Biomedical Press, Amsterdam I8 de Wardener. H E. and MacGregor. G. A (1983) Medicine (Baltimore) 62. 3 IO-326 19 Blaustein, M. P. and Hamlyn, J. M. (1983) Ann Intern Med. 98. Part 2 785-192 20 Ross, E. J. and Christie, S. B. M ( 1969) MedC tine (Baltimore) 48. 441-473 21 DeBold, A J.. Bornstein, H. B,, Veress A T. and Sonnenberg H. A (I 98 I) Life Sci 28,8994 22 Currie. M. G., Geller, D. M.. Cole, B. R. BoyIan, J. G., YuSheng W., Holmberg S. W. and Needleman. P. (1983) Science 22 I, 7 l-73 23 Genesf J. (1983) Ann Intern Med 98 (Part2). 744-749 JOHN MORDECAI
M.
HAMLYN
P. BLAUSTEIN
Depument of Ph.vsioiogy, University of Maryland School of Medicine, Baltimore MaNland 21201, USA.
ANNOUNCEMENT The First International Workshopon Neuroimmunomodulation(interactions among the nervous, immune and endocrine systems) will be held at the National Library of Medicine, N IH, Bethesda, MD, USA, on27-30 November 1984. All sessions will be plenary. This is the first of a continuing series that will take place in various countries once every two years. Proceedings will be published soon after each conference. Representatives from 15 or more countries are expected to participate. While the meeting is open, the workshop character severefy limits the possible number of registrants. In addition to the invited speakers, approximately25 “volunteer” papers will be accepted, and a maximum of 25 observers will be permitted. For further information, please contact Novera Herbert Spector, Ph.D. Fundamental Neurosciences Program NINCDS, NIH Federal Building, Room 916 Bethesda, MD., 20205
I984
oocyte, but these chains are assembled into complete AChRs that are inserted into the surface membrane where they function normally. Cells maintained in culture- for example. the neuroblastoma cell line Nl8 which lacks AChRs - can be used for microinjection experiments instead of oocytesU.‘-‘. For many experiments oocytes are preferable because they are so large (almost 1mm in diameter) and have such a large number of ribosomes: injection of a single oocyte can produce large enough quantities of protein to be easily detected by antibodies and used for some biochemical experiments. Cultured cells, however. are superior for some cell biological experiments (e.g. how does altered protein structure affect assembly and insertion of a channel) and possibly for physiological experiments (e.g. single channel record ing). For example. oocytes might in some circumstances yield too low a surface density to be useful for single channel recording but one could study, in principle at leasf just one AChR copy in a cultured neuron by using the whole cell recording configuration (see Ref. 14). In summary. then, the relationship between AChR structure and function can be investigated by altering the genetic blueprint for the protein, having this blueprint expressed in cells, and studying the functional implications of alterations in the modified proteins. This method was described here in the context of a specific
307 example, but clearly the approach is a general one. Other channel types. recep tors and enzymes involved in second messenger systems (e.g cyclic nucleotides). cell surface proteins that participate in cell-cell recognition, mechanisms of pro tein sorting and insertion into the surface membrane, and proteins responsible for exocytotic events and membrane recovery can all, in principle. be studied in a precisely analogous way. Although the method is a powerful one, a number of complications are evident. First, the DNA specifying a protein to be studied must have been cloned and sequenced; so far, the only channel protein for which this is true is the AChR Next. the manipulations involved can be rather time consuming with several weeks of work needed for making a single modification. Finally, the interpretation of the results is not at all automatic. As with other methods. several different tests of each hypothesis. by making various mod5 cations in the protein structure, will be required and predictions about functional implications of the changes made will have to be made with care and imagination. In conjunction with other approaches. though. site directed mutagenesis should provide a key for unravelling the molecular basis for nervous system function. Reading list 1 Kao. P.N.. Silver. M.L.
Dwork. A.J.. Kaldany. R-RJ.. Wideman J.. Stein S. and Karlin.
10
II I2 13 14
A (1984) J BiaL Chem 259. (in press) Walker. J. W.. Lukas, R J. and McNamee. M. G. ( I 98 I ) BiochemisfqJ 20. 2 19 I-2 I99 Dionne. V. E.. Steinback J. H. and Stevens. C. F. ( 1978)J. PhysioL {London) 28 I. 42 1444 Noda. M., Takahashi. H, Tanabe. T.. Toyosato. M. Furutani. Y.. Hirose. T., Asai. M.. Inayama. S.. Miyata. T. and Numa S. (I 982) Nature (London) 299, 793-797 Winter. G., Fersht A R. Wilkinson A. J.. Zoller. M. and Smith. M. ( 1982) Nature (Low don) 299.156-158 Green M. R. Mania& T. and Melton. D. A. (1983) Cell 32,681-694 Mom& S. M.. Pettersson. I.. Hinterberger. M.. Karmas, A. and Steitl J. A (1983) Cell 33. 509-5 18 Sumikawa. K.. Houghton. M.. Emtage. J. S.. Richards, B. M. and Barnard. E. A. (1981) Nature (London) 292. 862-864 Barnard. E. A. Miledi. R and Sumikawa K (1982) Proc R Sot. London B215.241-246 Mishina. M., Kurosaki. T.. Tobimatsu. T.. Morimoto. Y.. Noda. M.. Yamamoto. T.. Terao. M.. Lindstrom. J.. Takahashi. T.. Kuno. M. and Numa. S. ( 1984) Nature (London) 307.604608 Gurdon. J. B. and Melton. D. A ( I98 I ) Annu Res Gener 15. 189-218 Graessmann A.. Graessman. M. and Mueller. C. ( 1980) Methods Enz.vmoL 65. 8 16-825 Stacey. D. W. and Allfrey. V. G. (1916) Ce119. 725-732 Sakmann B. and Neher, E. ( 1983) in Single Channel Recording, Plenum Press. New York CHARLES
F. STEVENS
Section ofMolecularNeurobiolog.x Yale Universir?, School of Medicine. 333 Cedar SrreeC New Haven. CT 06510. USA.
induce positive inotropic effects in hearts or inhibit the short circuit current in amphibian epit.helium’O. Many investigators have assumed that an extract is digitalis-like if it produces one or more of these effects. However, many substances The quest for endogenous digitalislike factors has been given new impetus by the that are not inhibitors of the Na’ pump fmding that theplasma activity of theseputativefactors mayparticipate in the renal may appear to have‘digitalis-like’ propercontrol ofjluid and electrolyte metabolism and may play a role in the etiology of ties, as judged from their ability to produce hypertension The brain may be intimatelv involved in controlling theactivity ofthese cardiotonic effects or inhibit trans-epithe lial sodium transport Conversely, some factors. inhibitors of the Na’ pump, such as Ca” The discovery that enkephalins are endo exchange mechanism’. or vanadate, would not be considered genous opiates has accelerated the search There has been much speculation about ‘digitalis-like’. for other endogenous factors in mammalian the existence of endogenous factors with Efforts to isolate substances with DLA systems where specific receptors with ‘digitalislike’ activity (DLA) since Schatz- have generally begun with large quantities high affinity for exogenous substances, mamY first described the inhibition of of source material from humans or animals have been identified A particularly good active monovalent cation transport in red (plasma or urine, or homogenates of cells by digitalis glycosides. Examples of various tissues, e.g brain kidneys, adrenals, example is the interaction of the digitalis glycosides and other cardiotonic steroids ‘digitalis-like’ properties include: (a) in- or amphibian skin), often under different with the sodium pump. The digitalis glyco hibition of ouabainsensitive cellular ca- physiological conditions. In most cases, sides have been used for two centuries to tion fluxes3.? (b) inhibition of( Na’ i- K’) extracts have been chromatographed and treat congestive heart failure. Their -ATPase activity in isolated plasma mem- concentrated to increase DLA, which is mechanism of action is uncertain, but is brane preparation9.6.7.8; (c) competition usually quite low in the starting materials. believed to involve the binding to, and between radiolabelled digitalis glycosides However, these procedures may also amp subsequent inhibition of, the cardiac sar- and putative ‘digitalis-like’ factors, for lify the activities of interfering substances, either membrane-associated binding colemmal sodium pump; this indirectly often in a non-linear manner, so that they increases cardiac contractility by promotsites6.8.9or antibodies raised against specific are then mistaken for the substance(s) of digitalis glycosides6; and (d) ability to interest Furthermore, both the bioassays ing calcium accumulation via a Na’/CaZ’
Endogenous digitalis-like compounds: putative regulators of the sodium pump
0
19H4. Elrcwc :r Saencc
Pubhcatums
B.V.. Amsterdam
0.178 - 5Y I2/84/S02.00
TINS - September 1984
308 and biochemical methods that are fre quently used to detect DLA are, themselves, subject to many types of interference. These may be some of the reasons why no endogenous digitalislike sub stances have been isolated and characterized in the 30 years since the discovery of the interaction between the digitalis glyco sides and the Na’ pump, and the 15 years since the first report of an endogenous inhibitor of (Na’ + K’)-ATPase. The purification of digitalislike substances will obviously depend upon the establish ment of reliable assays, that can provide predictable results when several of the criteria mentioned above are tested. Since the pioneering observations of de Wardener and Mills”, numerous investigators have convincingly demonstrated that extracellular fluid (ECF) volume expansion triggers a natriuresis that can not be explained by modulation of aldo sterone secretion, or alteration of glomeru lar filtration rate. Extracts of plasma or urine from volume-expanded animals and man were found to contain one or more factors with DLA, as identified by the aforementioned criteria For example, a natriuretic factor was found in the urine of salt-retaining (volume-expanded) uremic patients, but not in that of salt-losing nephrotics I2. Furthermore, there is evidence that plasma from uremic patients contains elevated levels of a circulating inhibitor of Na’ pumpsL3J4. This substance should have a natriuretic effect analogous to that produced by digitalis glycosides, that can be explained by inhibition of renal tubule Na+ pumps - especially in the distal tubule’4, where the final composition of the urine is determined. Dahl15 was the first to suggest that a circulating natriuretic factor might be involved in the etiology of hypertension. Later workers postulated that this natriuretic, hypertensinogenic hormone might be a Na+ pump inhibitor, and that it might play a central role in the genesis of some ‘volumedependent’ forms of experimental hypertensionI and of essential hypertension in man”~ 18.Recent direct evidence shows that patients with essential hypertension have elevated plasma levels of a circulating Na’ pump inhibito?.‘. This substance could promote the rise in blood pressure by providing increased amounts of Ca*+, as well as Na+, to vascular smooth muscle and sympathetic neurones via a Na+/Caz+ exchange mechanism17~‘g. Numerous animal experiments indicate that the brain has a key role in controlling the activity of this natriuretic agent In rats, lesions in the anteroventral region of the hypothalamus adjacent to the third ventricle (AV3V), abolish the secretion
of a Na” transport inhibitor that usually is present in the plasma in mineralocorticoid (plus salt)-induced hypertension4, and foL lowing ECF volume expansionlO. These AV3V lesions also lead to hypematremia, and they attenuate or alleviate the rnineralo corticoid-induced and other types of saltdependent hypertension The rare human syndrome, ‘essential hypematremia’, that is usually associated with damage to the hypothalamic region of the brainzO, may be a consequence of impaired secretion of a digitalis-like material with natriuretic and hypertensinogenic properties19. Further evidence that the regulation of salt and water balance is far more complex than previously suspected comes from the discovery of several (cardiac) atria1 pep tides with natriuretic activity2 ‘. However, these substances cause vasodilation**, and do not inhibit (Na’ + K+)-ATPase23, therefore they are not digitalis-like. The convergence of these multiple lines of evidence, along with the newly-recognized pathophysiological implications, have helped to generate excitement about the role of digitalis-like substances in cation transport, and salt and water metabolism The key to unravelling these fundamental mechanisms is the isolation and characterization of the endogenous digitalislike compounds. Reading list I Baker. P. F., Blaustein M. P.. Hodgkin A L and Steinhardt. R A ( 1969)J Ph.vsioL (London) 200.497-527 2 Schatzmann. H. J. (1953) Helv. PhysioL Pharmacol Acru 11. 346-354 3 Poston, L., Sewell R B.. Wilkinson S. P.. Richardson, P. J., Williams, R. Clarkson, E. M, MacGregor. E. A and dewardener, H. E. (1982)Br. Mea! J. 282.847-849 4 SonguMize, E., Bealer. S. L and Caldwell. R W. (1982) HJpertension 4,575-580 5 Gonick. H. C., Kramer, H. J., Paul, W. and Lu, E. (1977) Clin Sci MoL Med 53, 329-334 6 Gruber, K. A. Whitaker, J. M. and Buckalew, V. M.. Jun (1980) Nature(London) 237,143145
-
7 Hamlyn J. M, Ringel. R. Schaeffer. J., Levinson, P. D.. Hamilton B. P.. Kowarski. A A and Blaustein, M. P. (1982) Nature. London 300.650-652 8 Shimoni. Y., Gotsman. M.. Deutsch, J.. Kachalsky. S. and Lichtstein D. ( 1984) Nature (London) 307. 369-371 9 Devynck M. A., Pemollet. M G., Rosenfeld, J. B. and Meyer, P. (1983) BI: Med J. 287. 631-634 IO Bealer, S. L. Haywood J. R. Gruber, K. A. Buckalew, V. M. Jun., Fink, G. D.. Brody. M. J. and Johnson. A. K (1983) Am .L PhysioL 244. R5l-R57 I I de Wardener, I-I E., Mills, I. H.. Clapham. W. F. and Hayter. C. J. ( I96 I ) C/in. Sci 2 I. 249-258 12 Bourgoignie. J. J., Hwang, K H., Ipakchi, E. and Bricker, N. S. (1974) J. Clin Invest 53. 1559-1567 I3 Smith, E. K M. and Welt. L G. (1970) Arch Intern Mea! 126.827-830 I4 Fine, L G.. Bourgoignie, J. J.. Hwang K. H. and Bricker, N. S. (1976) J. Ciin Invest 58. 590-597 I5 Dahl, L K.. Knudsen, D. K D. and lwai J. (1969) Circ. Reseurch (Suppl r). I-21 - I-33. 24-25 I6 Haddy, F. J. and Overbeck. H. W. ( 1976) Life Sci 19.935-948 17 Blaustein, M. P. ( 1977) in Excitation-Contmction Coupling in Smooth Muscle (Casteels, R, Godfraind T. and Ruegg, J. C., eds), pp. IOI108. Elsevier/North Holland Biomedical Press, Amsterdam I8 de Wardener. H E. and MacGregor. G. A (1983) Medicine (Baltimore) 62. 3 IO-326 19 Blaustein, M. P. and Hamlyn, J. M. (1983) Ann Intern Med. 98. Part 2 785-192 20 Ross, E. J. and Christie, S. B. M ( 1969) MedC tine (Baltimore) 48. 441-473 21 DeBold, A J.. Bornstein, H. B,, Veress A T. and Sonnenberg H. A (I 98 I) Life Sci 28,8994 22 Currie. M. G., Geller, D. M.. Cole, B. R. BoyIan, J. G., YuSheng W., Holmberg S. W. and Needleman. P. (1983) Science 22 I, 7 l-73 23 Genesf J. (1983) Ann Intern Med 98 (Part2). 744-749 JOHN MORDECAI
M.
HAMLYN
P. BLAUSTEIN
Depument of Ph.vsioiogy, University of Maryland School of Medicine, Baltimore MaNland 21201, USA.
ANNOUNCEMENT The First International Workshopon Neuroimmunomodulation(interactions among the nervous, immune and endocrine systems) will be held at the National Library of Medicine, N IH, Bethesda, MD, USA, on27-30 November 1984. All sessions will be plenary. This is the first of a continuing series that will take place in various countries once every two years. Proceedings will be published soon after each conference. Representatives from 15 or more countries are expected to participate. While the meeting is open, the workshop character severefy limits the possible number of registrants. In addition to the invited speakers, approximately25 “volunteer” papers will be accepted, and a maximum of 25 observers will be permitted. For further information, please contact Novera Herbert Spector, Ph.D. Fundamental Neurosciences Program NINCDS, NIH Federal Building, Room 916 Bethesda, MD., 20205