104
BIOCHEMICAL SOCIETY TRANSACTIONS
fraction was described (Wisher & Evans, 1975) as originating from the lateral face of the hepatocyte, and as such would be expected to bind only small quantities of ligands. A vesicular plasma-membrane fraction (density 1.13 g/ml), having high specificactivities of the marker enzymes 5‘-nucleotidase and alkaline phosphodiesterase, and previously described as being derived from the bile-canalicular face, also bound only small quantities of the ligands. Combined histochemical and radioautographic studies showed radioactivity in close proximity to the blood-sinusoidal surface of hepatocytes in livers perfused with iodinated wheat-germ agglutinin. However, associated with larger blood vessels was some unbound radioactivity which was not removed after extensive perfusion. This apparently unbound lectin may be able to attach itself to plasma-membrane material after tissue homogenization and could account for the low amount of radioactivity associated with the bile-canalicular and lateral fractions. It is unlikely that ligand associated with these fractions is due to redistribution within the plasma membranes, since perfusions were for short duration and at low temperature. Centrifugation of plasma-membrane subfractions on sucrose density gradients showed that the peaks of 5‘-nucleotidase and alkaline phosphodiesterase activity were coincident and were associated with the major plasma-membrane bands of both ‘heavy’ and ‘light’ fractions. Although the major peak of lectin and glucagon binding was also associated with the major band of plasma membrane, there appeared to be a marked heterogeneity in the distribution of lectin and glucagon binding in ’heavy’ plasma-membrane subfractions. The highest specific radioactivity of glucagon binding was associated with plasma membrane of density 1.11-1.13g/ml, whereas that of wheat-germ agglutinin coincided with the major bands of plasma membrane at densities 1.11-1.13 and 1.161.17g/ml. In summary, the use of iodinated wheat-germ agglutinin and glucagon has now permitted the direct identification of plasma-membrane subfractions derived from the blood-sinusoidal surface of the hepatocyte. The ligand-binding properties of a vesicular fraction, which had high activities of 5‘-nucleotidase and alkaline phosphodiesterase, was consistent with the cytochemical evidence of Essner et al. (1958) that this fraction was derived from the bile canaliculus. Chang, K. W., Bennett, V. & Cuatrecasas, P. (1975) J. Biol. Chem. 250,488-500 Essner, E., Novikoff,A. B. & Masek, B. (1958) J . Biophys. Biochem. Cytol. 4,711-716 Farquhar, M. G., Bergeron, J. J. M. & Palade, G. E. (1974) J. Cell Biol. 60,8-25 Hunter, W. M. & Greenwood, F. C. (1962) Nature (London)194,495-496 Wisher, M. H. & Evans, W. H. (1975) Biochem. J. 146, 375-388
The Relationship between Calcium Ion Gates and the Stimulation of Phosphatidylinositol Turnover SHAMSHAD S. JAFFERJI and ROBERT H. MLCHELL Department of Biochemistry, University of Birmingham, P.O. Box 363, Birmingham B15 2iT, W.K.
Agents which stimulate the contractile response of ileum smooth muscle and the secretory response of the exocrine pancreas bring about a stimulus-response coupling sequence which is mediated through a rise in intracellular Ca2+concentration (Hurwitz & Suria, 1971; Douglas, 1974): this rise in intracellular Ca2+concentration is at least partially due to an increase in cell-surface Caz+permeability (i.e. to the opening of cellsurface Caz+gates). The same stimuli also cause an increase in phosphatidylinositol turnover, as measured by the incorporation of [32P]Piinto phosphatidylinositol (L. E. Hokin, 1968; M.R. Hokin, 1968a,b; Jafferji & Michell, 1976u,b,c). The longitudinal smooth muscle of guinea-pig ileum possesses receptors for muscarinic cholinergic and H I-histaniinergic stimuli and for 5-hydroxytryptamine, all of which initiate contraction through an increase in cell-surface Caz+permeability (Triggle, 1971). This tissue also possesses potential-sensitive CaZ+gates which can be opened by elevation 1977
566th MEETING, CAMBRIDGE
105
Table 1 . Responses of ileum smooth muscle and exocrine pancreas to various stimuli
Tissue Ileum smooth muscle
Pancreas (exocrine)
Stimulus (type of receptor) Acetylcholine (muscarinic) Histamine (HI) 5-Hydroxytryptamine (methysergidesensitive) High extracellular [K+] Acetylcholine (muscarinic) Pancreozymin High extracellular [K+]
Stimulation of phosphatidylinositol turnover YeS
Stimulation of physiological response Yes (contraction)
Yes Yes
Yes (contraction) Yes (contraction)
Yes Yes
Yes (contraction) Yes (secretion)
Yes No
Yes (secretion) No (secretion)
of the extracellular K+ concentration (Hurwitz & Suria, 1971 ;Triggle, 1971 ;Berridge, 1975). These four stimuli all produced enhanced turnover of phosphatidylinositol, with the relevant receptors showing the same pharmacological characteristics as those which trigger contractility (Jafferji & Michell, 1976u,b,d; see Table 1). The physiological secretory response in the exocrine pancreas is controlled through muscarinic cholinergic receptors and receptors for pancreozymin; both types of receptors cause an increase in intracellular Cat+ concentration. Unlike ileum smooth muscle, the pancreas does not possess Cat+ gates that are opened by depolarization caused by elevation of extracellular K+ concentration (Matthews, 1974). In the pancreas phosphatidylinositol turnover is stimulated by muscarinic cholinergic stimuli and by pancreozymin (L. E. Hokin, 1968; M. R. Hokin, 1968u,b; S . S . Jafferji & R.H. Michell, unpublished work), but not by increasing the extracellular K+ concentration (Jaffeji & Michell, 1976d) (see Table 1 ) . It is clear from these studies that stimulated phosphatidylinositol turnover cannot be implicated in the ultimate contractile or secretory responses of these two tissues, but that its function must be sought in some earlier feature common to both tissues. It has been shown that the increase in phosphatidylinositol turnover is not a consequence of the increase in intracellular Ca2+concentration brought about by receptor activation (See Michell et al., 1976; Jafferji & Michell, 1976c), and it therefore appears that its function is most likely to lie in the mechanisms involved in the control of cell-surface CaZ+ gates by stimuli (Michell, 1975; Michell et al., 1976; Jafferji & Michell, 1976a,b,c,d). This point of view is especially strongly supported by the correlation between the phosphatidylinositol response and potential-sensitive Cat+ gating in pancrease and in ileum smooth muscle. Bemdge, M. J. (1975) Ado. Cyclic Nucleotide Res. 6, 1-98 Douglas, W. W. (1974) Biochem. SOC.Symp. 39,l-28 Hokin, L. E. (1968) Znt. Rev. Cytol. 23, 187-208 Hokin, M. R. (1968~)Arch. Biochem. Biophys. 124,271-279 Hokin, M.R. (19686) Arch. Biochem. Biophys. 124,280-284 Hurwitz, K. & Suria, A. (1971) Annu. Rev. Phnrmacol. 11,303-326 JafTerji, S . S. & Michell, R. H. (1976~)Biochem. J. 154, 653-657 Jafferji, S. S. & Michell, R. H. (19766) Biochem. Pharmacol. 25, 1429-1430 Jderji, S. S. & Michell, R. H. (1976~)Biochem. J. 160, 163-169 Jafferji, S. S. & Michell, R. H. (1976d)Biochem. J. 160,397-399 Matthews, E. K . (1974) in Secretory Mechanisms of Exocrine Tissues (Thorn, N. A. & Petersen, 0. H., eds.), pp. 185-198, Munksgaard, Copenhagen Michell, R. H. (1975) Biochim. Biophys. Acta 415,81-147
Vol. 5
106
BIOCHEMICAL SOCIETY TRANSACTIONS
Michell, R. H., Jones, L. hi. & Jafferji, S. S . (1976) in Stimulus-Secretion CoupZing in the Gastrointestinal Tract (Case, R. M. & Goebell, H., eds.), pp. 89-103, MTP, Lancaster Triggle, D. J. (I 971) Neurotransmitter-Receptor Interactions, Academic Press, New York
Evolutionary and Seasonal Adaptation of Membranes to Temperature ANDREW R. COSSINS* Department of Physiology and Biophysics, University of Illinois, Urbana, ZL 61801, U.S.,4.
Despite large advances in the past decade in our understanding of the structure and function of biological membranes, little is known about the factors which are important in the maintenance of the bilayer in its optimal state. Temperature is an important and all-pervasive influence, with dramatic effects on the thermodynamic state of the membrane interior (Jacobson & Papahadjopoulos, 1975; Vanderkooi et al., 1974). It may be expected that organisms which are exposed to seasonal shifts in temperature would possess some homoeostatic mechanism to mitigate the effect of temperature changes. The synaptic complex is thought to be particularly sensitive to modification during thermal acclimatization (Prosser, 1973), and a preparation enriched in synaptosomes from the brain of goldfish acclimatized to 5, 15 or 25°C has been used to determine the adaptive ability of animal membranes. The viscosity of the membrane interior was determined by using the fluorescence-polarization technique (Shinitsky et al., 1971) with 1,6It was diphenylhexatriene as probe, and reported as the average rate of probe rotation (3). assumed that the rotational characteristics of the probe are limited by its hydrophobic environment (Shinitsky et al., 1971; Cogan et al., 1973; Andrich & Vanderkooi, 1976); thus increased rotational rates indicate a more fluid membranous environment. Fluorescence lifetime was measured by using a cross-correlation phase fluorimeter (Spencer & Weber, 1969). Acclimatization ofgoldfish to warmer temperatures resulted in a significant shift of the viscosity/temperature graph to higher temperatures; i.e. when measured at an intermediate temperature, the viscosity of synaptosomal preparation isolated from warm-acclimatized goldfish was greater than for cold-acclimatized goldfish. These differences were highly reproducible, indicating that the absolute viscosity is a highly regulated parameter. The correlation between synaptosomal-membrane viscosity and cell-acclimatization temperature may be successfully extrapolated to include other fish species from diverse thermal environments [arctic sculpin (0.5"C)and desert pupfish (34"C)I as well as small mammals (37°C). Thus interspecific differences in synaptosomalmembrane viscosity appear to be related to differences in cell temperature rather than other evolutionary factors. This represents a clear adaptation to temperature over the evolutionary time-scale, since goldfish are incapable of acclimatizing over the entire 037°C range (Prosser, 1973), just as arctic sculpins die at moderately high temperatures (A. R. Cossins & C. L. Prosser, unpublished work). Phospholipid liposomes prepared from 5°C- and 25°C-acclimatized goldfish brain synaptosomes and rat brain synaptosomes showed similar differences in viscosity/ temperature profiles as observed for thenative membrane. Theacclimatization-dependent differences may therefore be explained by biochemical modification of membrane phospholipids. Cholesterol content and cholesterol/phospholipidmolar ratios were unaffected by thermal acclimatization. Synaptosomal membranes of cold-acclimatizedgoldfish possessed somewhat higher proportions of unsaturated fatty acid compared with warm-acclimatized goldfish. In addition, rat membranes contained substantially more saturated fatty acids than goldfish membranes, owing mainly to decreased proportions of c 2 2 : 6 ~ 3and increased proportions of c16:0, Cls:oand ci8:lm9 fatty acids.
* Present address: A.R.C. Institute of Animal Physiology, Babraham, Cambridge CB2 4AT, U.K.
1977
BIOCHEMICAL SOCIETY TRANSACTIONS
fraction was described (Wisher & Evans, 1975) as originating from the lateral face of the hepatocyte, and as such would be expected to bind only small quantities of ligands. A vesicular plasma-membrane fraction (density 1.13 g/ml), having high specificactivities of the marker enzymes 5‘-nucleotidase and alkaline phosphodiesterase, and previously described as being derived from the bile-canalicular face, also bound only small quantities of the ligands. Combined histochemical and radioautographic studies showed radioactivity in close proximity to the blood-sinusoidal surface of hepatocytes in livers perfused with iodinated wheat-germ agglutinin. However, associated with larger blood vessels was some unbound radioactivity which was not removed after extensive perfusion. This apparently unbound lectin may be able to attach itself to plasma-membrane material after tissue homogenization and could account for the low amount of radioactivity associated with the bile-canalicular and lateral fractions. It is unlikely that ligand associated with these fractions is due to redistribution within the plasma membranes, since perfusions were for short duration and at low temperature. Centrifugation of plasma-membrane subfractions on sucrose density gradients showed that the peaks of 5‘-nucleotidase and alkaline phosphodiesterase activity were coincident and were associated with the major plasma-membrane bands of both ‘heavy’ and ‘light’ fractions. Although the major peak of lectin and glucagon binding was also associated with the major band of plasma membrane, there appeared to be a marked heterogeneity in the distribution of lectin and glucagon binding in ’heavy’ plasma-membrane subfractions. The highest specific radioactivity of glucagon binding was associated with plasma membrane of density 1.11-1.13g/ml, whereas that of wheat-germ agglutinin coincided with the major bands of plasma membrane at densities 1.11-1.13 and 1.161.17g/ml. In summary, the use of iodinated wheat-germ agglutinin and glucagon has now permitted the direct identification of plasma-membrane subfractions derived from the blood-sinusoidal surface of the hepatocyte. The ligand-binding properties of a vesicular fraction, which had high activities of 5‘-nucleotidase and alkaline phosphodiesterase, was consistent with the cytochemical evidence of Essner et al. (1958) that this fraction was derived from the bile canaliculus. Chang, K. W., Bennett, V. & Cuatrecasas, P. (1975) J. Biol. Chem. 250,488-500 Essner, E., Novikoff,A. B. & Masek, B. (1958) J . Biophys. Biochem. Cytol. 4,711-716 Farquhar, M. G., Bergeron, J. J. M. & Palade, G. E. (1974) J. Cell Biol. 60,8-25 Hunter, W. M. & Greenwood, F. C. (1962) Nature (London)194,495-496 Wisher, M. H. & Evans, W. H. (1975) Biochem. J. 146, 375-388
The Relationship between Calcium Ion Gates and the Stimulation of Phosphatidylinositol Turnover SHAMSHAD S. JAFFERJI and ROBERT H. MLCHELL Department of Biochemistry, University of Birmingham, P.O. Box 363, Birmingham B15 2iT, W.K.
Agents which stimulate the contractile response of ileum smooth muscle and the secretory response of the exocrine pancreas bring about a stimulus-response coupling sequence which is mediated through a rise in intracellular Ca2+concentration (Hurwitz & Suria, 1971; Douglas, 1974): this rise in intracellular Ca2+concentration is at least partially due to an increase in cell-surface Caz+permeability (i.e. to the opening of cellsurface Caz+gates). The same stimuli also cause an increase in phosphatidylinositol turnover, as measured by the incorporation of [32P]Piinto phosphatidylinositol (L. E. Hokin, 1968; M.R. Hokin, 1968a,b; Jafferji & Michell, 1976u,b,c). The longitudinal smooth muscle of guinea-pig ileum possesses receptors for muscarinic cholinergic and H I-histaniinergic stimuli and for 5-hydroxytryptamine, all of which initiate contraction through an increase in cell-surface Caz+permeability (Triggle, 1971). This tissue also possesses potential-sensitive CaZ+gates which can be opened by elevation 1977
566th MEETING, CAMBRIDGE
105
Table 1 . Responses of ileum smooth muscle and exocrine pancreas to various stimuli
Tissue Ileum smooth muscle
Pancreas (exocrine)
Stimulus (type of receptor) Acetylcholine (muscarinic) Histamine (HI) 5-Hydroxytryptamine (methysergidesensitive) High extracellular [K+] Acetylcholine (muscarinic) Pancreozymin High extracellular [K+]
Stimulation of phosphatidylinositol turnover YeS
Stimulation of physiological response Yes (contraction)
Yes Yes
Yes (contraction) Yes (contraction)
Yes Yes
Yes (contraction) Yes (secretion)
Yes No
Yes (secretion) No (secretion)
of the extracellular K+ concentration (Hurwitz & Suria, 1971 ;Triggle, 1971 ;Berridge, 1975). These four stimuli all produced enhanced turnover of phosphatidylinositol, with the relevant receptors showing the same pharmacological characteristics as those which trigger contractility (Jafferji & Michell, 1976u,b,d; see Table 1). The physiological secretory response in the exocrine pancreas is controlled through muscarinic cholinergic receptors and receptors for pancreozymin; both types of receptors cause an increase in intracellular Cat+ concentration. Unlike ileum smooth muscle, the pancreas does not possess Cat+ gates that are opened by depolarization caused by elevation of extracellular K+ concentration (Matthews, 1974). In the pancreas phosphatidylinositol turnover is stimulated by muscarinic cholinergic stimuli and by pancreozymin (L. E. Hokin, 1968; M. R. Hokin, 1968u,b; S . S . Jafferji & R.H. Michell, unpublished work), but not by increasing the extracellular K+ concentration (Jaffeji & Michell, 1976d) (see Table 1 ) . It is clear from these studies that stimulated phosphatidylinositol turnover cannot be implicated in the ultimate contractile or secretory responses of these two tissues, but that its function must be sought in some earlier feature common to both tissues. It has been shown that the increase in phosphatidylinositol turnover is not a consequence of the increase in intracellular Ca2+concentration brought about by receptor activation (See Michell et al., 1976; Jafferji & Michell, 1976c), and it therefore appears that its function is most likely to lie in the mechanisms involved in the control of cell-surface CaZ+ gates by stimuli (Michell, 1975; Michell et al., 1976; Jafferji & Michell, 1976a,b,c,d). This point of view is especially strongly supported by the correlation between the phosphatidylinositol response and potential-sensitive Cat+ gating in pancrease and in ileum smooth muscle. Bemdge, M. J. (1975) Ado. Cyclic Nucleotide Res. 6, 1-98 Douglas, W. W. (1974) Biochem. SOC.Symp. 39,l-28 Hokin, L. E. (1968) Znt. Rev. Cytol. 23, 187-208 Hokin, M. R. (1968~)Arch. Biochem. Biophys. 124,271-279 Hokin, M.R. (19686) Arch. Biochem. Biophys. 124,280-284 Hurwitz, K. & Suria, A. (1971) Annu. Rev. Phnrmacol. 11,303-326 JafTerji, S . S. & Michell, R. H. (1976~)Biochem. J. 154, 653-657 Jafferji, S. S. & Michell, R. H. (19766) Biochem. Pharmacol. 25, 1429-1430 Jderji, S. S. & Michell, R. H. (1976~)Biochem. J. 160, 163-169 Jafferji, S. S. & Michell, R. H. (1976d)Biochem. J. 160,397-399 Matthews, E. K . (1974) in Secretory Mechanisms of Exocrine Tissues (Thorn, N. A. & Petersen, 0. H., eds.), pp. 185-198, Munksgaard, Copenhagen Michell, R. H. (1975) Biochim. Biophys. Acta 415,81-147
Vol. 5
106
BIOCHEMICAL SOCIETY TRANSACTIONS
Michell, R. H., Jones, L. hi. & Jafferji, S. S . (1976) in Stimulus-Secretion CoupZing in the Gastrointestinal Tract (Case, R. M. & Goebell, H., eds.), pp. 89-103, MTP, Lancaster Triggle, D. J. (I 971) Neurotransmitter-Receptor Interactions, Academic Press, New York
Evolutionary and Seasonal Adaptation of Membranes to Temperature ANDREW R. COSSINS* Department of Physiology and Biophysics, University of Illinois, Urbana, ZL 61801, U.S.,4.
Despite large advances in the past decade in our understanding of the structure and function of biological membranes, little is known about the factors which are important in the maintenance of the bilayer in its optimal state. Temperature is an important and all-pervasive influence, with dramatic effects on the thermodynamic state of the membrane interior (Jacobson & Papahadjopoulos, 1975; Vanderkooi et al., 1974). It may be expected that organisms which are exposed to seasonal shifts in temperature would possess some homoeostatic mechanism to mitigate the effect of temperature changes. The synaptic complex is thought to be particularly sensitive to modification during thermal acclimatization (Prosser, 1973), and a preparation enriched in synaptosomes from the brain of goldfish acclimatized to 5, 15 or 25°C has been used to determine the adaptive ability of animal membranes. The viscosity of the membrane interior was determined by using the fluorescence-polarization technique (Shinitsky et al., 1971) with 1,6It was diphenylhexatriene as probe, and reported as the average rate of probe rotation (3). assumed that the rotational characteristics of the probe are limited by its hydrophobic environment (Shinitsky et al., 1971; Cogan et al., 1973; Andrich & Vanderkooi, 1976); thus increased rotational rates indicate a more fluid membranous environment. Fluorescence lifetime was measured by using a cross-correlation phase fluorimeter (Spencer & Weber, 1969). Acclimatization ofgoldfish to warmer temperatures resulted in a significant shift of the viscosity/temperature graph to higher temperatures; i.e. when measured at an intermediate temperature, the viscosity of synaptosomal preparation isolated from warm-acclimatized goldfish was greater than for cold-acclimatized goldfish. These differences were highly reproducible, indicating that the absolute viscosity is a highly regulated parameter. The correlation between synaptosomal-membrane viscosity and cell-acclimatization temperature may be successfully extrapolated to include other fish species from diverse thermal environments [arctic sculpin (0.5"C)and desert pupfish (34"C)I as well as small mammals (37°C). Thus interspecific differences in synaptosomalmembrane viscosity appear to be related to differences in cell temperature rather than other evolutionary factors. This represents a clear adaptation to temperature over the evolutionary time-scale, since goldfish are incapable of acclimatizing over the entire 037°C range (Prosser, 1973), just as arctic sculpins die at moderately high temperatures (A. R. Cossins & C. L. Prosser, unpublished work). Phospholipid liposomes prepared from 5°C- and 25°C-acclimatized goldfish brain synaptosomes and rat brain synaptosomes showed similar differences in viscosity/ temperature profiles as observed for thenative membrane. Theacclimatization-dependent differences may therefore be explained by biochemical modification of membrane phospholipids. Cholesterol content and cholesterol/phospholipidmolar ratios were unaffected by thermal acclimatization. Synaptosomal membranes of cold-acclimatizedgoldfish possessed somewhat higher proportions of unsaturated fatty acid compared with warm-acclimatized goldfish. In addition, rat membranes contained substantially more saturated fatty acids than goldfish membranes, owing mainly to decreased proportions of c 2 2 : 6 ~ 3and increased proportions of c16:0, Cls:oand ci8:lm9 fatty acids.
* Present address: A.R.C. Institute of Animal Physiology, Babraham, Cambridge CB2 4AT, U.K.
1977
