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94 Use of histochemistry in pharmacological studies
29
Abstracts of Pafers 93
Methods
for the Study of Pharmacological Effects at Cellular and SubcelInlar Levels. Introductory Remarks. 0. 11. LOWRY pJ.S.A.).
94
Use of Histochemistry in Pharmacological Studies. G. B. KOELLE(U.S.A.). The chief obstacle to the localization of sites of drug action at the cellular level is our present ignorance concerning the nature of the groups with which most drugs react to produce their characteristic pharmacological effects, i.e. the receptors. Demonstration of the distribution of a drug does not by itself permit distinction betwtxn critical active sites and non-specific storage sites. In a few instances (e.g. anticholinesterase (anti-ChE) agents, carbonic anhydrasc inhibitors), the significant tissue component affected is known with reasonable cc‘rtainty, and in others (c.g. monoamixie nxidasc inhibitors, mercurial diuretics), potential, but not necessarily the critical, cellular constituents have When this information is available, been identified. h~tochemical demonstration or In~croch~:lnicaI determination of changes in the reactivity of the active group or enzyme produced by the drug may provide more direct information on its site of action. This? of course. does not exclude the possibility of its total pharmacological effect being the result of reaction with more than one type of receptor. Concrete examples of the foregoing principles are provided by histochemical investigations of the sites of action of anti-ChE agents. Such studies have shown that acetylcholinesterase (AChE) may be oriented cxtcrnally or internally with respect to the cell membrane, and that certain pharmacological effects are produc4 by inhibition of only the external AChE. In addition, they have provided evidence of a dual role of acetylcholine [ACh) in synaptic transmission : an action at the presynaptic site, to promote the liberation of more quanta of ACh or of other transmitters, and, in the case of choiinergic neurons, at the postsynaptic site.
95 Contribution of Eleetromnicroscopy to NeuroE. DEROBERTIS pharmacological Problems. (.hrgentina). New ultra-structural details of the synaptic region---in addition to the synaptic vesicles(l’--will be described in isolated nerve endings and in sections of the brain cortex. These include a system of intersynaptic filaments and a subsynaptic web forming a tight complex with both synaptic membranes. The possible significance of these two new structures and the physiological role of synaptic vesicles will be discussed. The so-called “mitochondrial fraction” of the brain has been subdivided into five subfractions, one containing my&n, another only mitochondria, a tbird containing broken nerve endings and synaptic complexes, and two formed by rather intact
nerve endings which represent about 15 per cent of total brain weight. The AChE and SDH activities and the ACh content of these fractions was determined. The resultssuggest the probable existence of a different population of cholinergic and non-chdinergic endings in the two fractions. These results also throw some light on the localization of AChE in central synapses. The ultra-structure of adrenergic nerve libres and endings with the presence of a specific plurivesicular A structurally similar material will be described. plurivesicular component is found in the secretory processes of the pineal gland. Pharmacoiogical and other experimental studies suggest that thiq gland is formed by adrenergic neurosecretory cell5 and that the plurivesicrrlar material is the site of’ storage of 5_hydroxytryptamine, catecholamines and other biogenic amines secreted by the pineal gland. These and other results will be interpreted within an unified neurosrcretory theory that embodies synaptic processes and neurosecretion. The contribution of several of our collaborators in these studies will be acknowledged.
1. 1,~ ROBERTIS
and
BENNETT (1954).
96 Some Morphological and Biochemical Aspects of the Sarcotobular System in Frog Skeletal and Muscle Cell. E. :iNDERSSON-‘&DERGREN U. MIXATELLO (Sweden). One of the main components ol’ the skeletal muscle cell is a system of tubular and vesicular structures localized in between the m~~o~brils. From the morphological distribution and characteristic organization of the sarcotubules”, 2’ and from information gained from experiments by local electrical stimulation(3~ the conclusion has been drawn that these sarcotubules could be responsible for thr propagation of the stimulating impulse from the ccl1 membrancb inwards to the contractile By means of differential high speed material. centrifugation this system of sarcotubular elements has been isolated. ‘l‘he isolated fraction from frog muscle homogenate contains small tubular and vesicular elements and small particles not attached to the membranes.‘*’ The sedimentation, biochemical and morphological data of the sarcotubular fraction show similarity to the properties of the so-called relaxing The relaxing activity factor described by Marsh.f5’ of the sarcotubular fraction is shown by its capacity to inhibit the myofibriilar ATP-ase and the myofibrillar superprecipitation induced by ATP. The activity seems to be localized to the membranous The components of the sarcotubular fraction. fractions of the muscle homogenate containing the mitochondrial fragments or the small particles do not show the same properties. The biochemical data of the sarcotubular fraction togrther with the morphological appearance of the
Abstracts of Pafers 93
Methods
for the Study of Pharmacological Effects at Cellular and SubcelInlar Levels. Introductory Remarks. 0. 11. LOWRY pJ.S.A.).
94
Use of Histochemistry in Pharmacological Studies. G. B. KOELLE(U.S.A.). The chief obstacle to the localization of sites of drug action at the cellular level is our present ignorance concerning the nature of the groups with which most drugs react to produce their characteristic pharmacological effects, i.e. the receptors. Demonstration of the distribution of a drug does not by itself permit distinction betwtxn critical active sites and non-specific storage sites. In a few instances (e.g. anticholinesterase (anti-ChE) agents, carbonic anhydrasc inhibitors), the significant tissue component affected is known with reasonable cc‘rtainty, and in others (c.g. monoamixie nxidasc inhibitors, mercurial diuretics), potential, but not necessarily the critical, cellular constituents have When this information is available, been identified. h~tochemical demonstration or In~croch~:lnicaI determination of changes in the reactivity of the active group or enzyme produced by the drug may provide more direct information on its site of action. This? of course. does not exclude the possibility of its total pharmacological effect being the result of reaction with more than one type of receptor. Concrete examples of the foregoing principles are provided by histochemical investigations of the sites of action of anti-ChE agents. Such studies have shown that acetylcholinesterase (AChE) may be oriented cxtcrnally or internally with respect to the cell membrane, and that certain pharmacological effects are produc4 by inhibition of only the external AChE. In addition, they have provided evidence of a dual role of acetylcholine [ACh) in synaptic transmission : an action at the presynaptic site, to promote the liberation of more quanta of ACh or of other transmitters, and, in the case of choiinergic neurons, at the postsynaptic site.
95 Contribution of Eleetromnicroscopy to NeuroE. DEROBERTIS pharmacological Problems. (.hrgentina). New ultra-structural details of the synaptic region---in addition to the synaptic vesicles(l’--will be described in isolated nerve endings and in sections of the brain cortex. These include a system of intersynaptic filaments and a subsynaptic web forming a tight complex with both synaptic membranes. The possible significance of these two new structures and the physiological role of synaptic vesicles will be discussed. The so-called “mitochondrial fraction” of the brain has been subdivided into five subfractions, one containing my&n, another only mitochondria, a tbird containing broken nerve endings and synaptic complexes, and two formed by rather intact
nerve endings which represent about 15 per cent of total brain weight. The AChE and SDH activities and the ACh content of these fractions was determined. The resultssuggest the probable existence of a different population of cholinergic and non-chdinergic endings in the two fractions. These results also throw some light on the localization of AChE in central synapses. The ultra-structure of adrenergic nerve libres and endings with the presence of a specific plurivesicular A structurally similar material will be described. plurivesicular component is found in the secretory processes of the pineal gland. Pharmacoiogical and other experimental studies suggest that thiq gland is formed by adrenergic neurosecretory cell5 and that the plurivesicrrlar material is the site of’ storage of 5_hydroxytryptamine, catecholamines and other biogenic amines secreted by the pineal gland. These and other results will be interpreted within an unified neurosrcretory theory that embodies synaptic processes and neurosecretion. The contribution of several of our collaborators in these studies will be acknowledged.
1. 1,~ ROBERTIS
and
BENNETT (1954).
96 Some Morphological and Biochemical Aspects of the Sarcotobular System in Frog Skeletal and Muscle Cell. E. :iNDERSSON-‘&DERGREN U. MIXATELLO (Sweden). One of the main components ol’ the skeletal muscle cell is a system of tubular and vesicular structures localized in between the m~~o~brils. From the morphological distribution and characteristic organization of the sarcotubules”, 2’ and from information gained from experiments by local electrical stimulation(3~ the conclusion has been drawn that these sarcotubules could be responsible for thr propagation of the stimulating impulse from the ccl1 membrancb inwards to the contractile By means of differential high speed material. centrifugation this system of sarcotubular elements has been isolated. ‘l‘he isolated fraction from frog muscle homogenate contains small tubular and vesicular elements and small particles not attached to the membranes.‘*’ The sedimentation, biochemical and morphological data of the sarcotubular fraction show similarity to the properties of the so-called relaxing The relaxing activity factor described by Marsh.f5’ of the sarcotubular fraction is shown by its capacity to inhibit the myofibriilar ATP-ase and the myofibrillar superprecipitation induced by ATP. The activity seems to be localized to the membranous The components of the sarcotubular fraction. fractions of the muscle homogenate containing the mitochondrial fragments or the small particles do not show the same properties. The biochemical data of the sarcotubular fraction togrther with the morphological appearance of the