Electron-microscopic studies on the relationship between the frequency of parathyroid storage granules and serum calcium levels in the rat

Summary In the rat parathyroid, the mean number of storage granules (NSG) per chief cell has been electron-microscopically studied and correlated with the mean serum calcium level (SCL). In animals given 4% CaCl₂ plus vitamin D₂ for 3 days, SCL is significantly elevated and NSG is increased. When these animals are injected with 2% EDTA, SCL is lowered to 8 mg/dl, but NSG is not affected; in those injected with 4% EDTA, however, SCL declines to a minimum (5.8 mg/dl) after 30 min, and NSG is also decreased. Control SCL are 8.9 mg/dl. These results indicate that storage granules may not be released until SCL is depressed to a certain level.In rats 3 weeks after castration, the chief cells show hyperplastic changes and SCL is at a low concentration (8.0 mg/dl). NSG, however, remains almost within control limits. Castrated animals injected with 4% EDTA show a hypocalcemia and a decrease in NSG, but NSG gradually recovers over a period of 6h. These data suggest that storage granules can be produced even under lower calcium concentrations. It is concluded that storage granules may be constantly produced and stored, and are released only as an emergency supply of hormone.     

Effects of short-term treatment with calcium on the parathyroid gland of the rat,under particular consideration of the alteration of storage granules

Summary Short-term effects of CaCl₂-treatment on parathyroid cells of the rat, especially on their storage granules, were studied at the ultrastructural level. After an injection of 4% CaCl₂, serum calcium levels (SCL) rapidly increased from 9.1 mg/dl (controls) to a maximum of 14.9 mg/dl at 20 min. At 5 min after the injection, the number of type-I storage granules (large core) [NSG-I] and that of type-II storage granules (small core) [NSG-II] remained unchanged, in spite of elevated SCL (12.4 mg/dl). As soon as SCL rose to 13.2 mg/dl at 7.5 min, NSG-I gradually decreased to a minimum at 30 min; in contrast, NSG-II gradually increased to a maximum at 30 min. Vacuolar bodies also increased together with the augmentation of type-II storage granules. The average diameter of the core of the storage granules decreased significantly after the injection. Protein A-gold method for immunocytochemistry showed that the cores of these granules contain parathormone. Acid-phosphatase activity was occasionally found in storage granules of both types, especially in those of type II. It is concluded (i) that type-I storage granules may be transformed into vacuolar bodies via type-II granules as a result of hydrolysis, and (ii) that these processes may be accelerated during hypercalcemia.     

Effects of pilocarpine treatment and of electrical stimulation of the vagus nerve on the rat parathyroid gland, with special reference to the alteration of storage granules

Effects of pilocarpine treatment and of electrical vagal stimulation on the rat parathyroid were studied ultrastructurally. The number of type I storage granules with a narrow halo (NSG-I) and that of type II storage granules having a wide halo (NSG-II) were calculated. After pilocarpine treatment, NSG-I gradually decreased and reached a minimum at 30 min; in contrast, NSG-II gradually increased and reached a maximum at 20 min, but thereafter it slightly decreased and instead vacuolar bodies increased. Excluding these alterations, the ultrastructure of parenchymal cells showed no remarkable changes. Electrical vagal stimulation furthermore confirmed these results. Acid phosphatase activity was occasionally found in storage granules of both types in control and experimental rats. It was concluded that storage granules normally may be transformed from type I into type II and finally into vacuolar bodies as a result of hydrolysis, and that these processes may be accelerated by parasympathetic stimulation.     

Regulation of glycogen metabolism in liver by the autonomic nervous system. VI. Possible mechanism of phosphorylase activation by the splanchnic nerve.

The effects of autonomic-nerve stimulation on the activities of phosphorylase (EC 2.4.1.1), dephospho-phosphorylase kinase (EC 2.7.1.38) and phosphorylase phosphatase (EC 3.1.3.17), and on the concentration of adenosine 3', 5'-monophosphate in rabbit liver were investiaged. Results were compared with the effects of epinephrine and glucagon on these enzymes. 1. The acitivity of liver phosphorylase increased rapidly and markedly on electrical stimulation of the splanchnic nerve, or after intraportal administration of epinephrine or glucagon. The activity was not affected by vagal stimulation. 2. The activity of dephospho-phosphorylase kinase increased about 2--3-fold 1 min after injections of epinephrine and glucagon, glucagon causing more activation than epinephrine. The enzyme activity was not altered by splanchnic-nerve, or vagal stimulation. 3. Injections of epinephrine and glucagon caused marked elevation of liver adenosine 3', 5'-monophosphate within a few minutes. With epinephrine, the nucleotide concentration rose to a maximum after 1 min and amounted to about 3-fold increase, while with glucagon the maximum increase of approximately 8-fold increase was observed after 2 min. Stimulation of the splanchnic nerve for 10 min did not affect the adenosine 3', 5'-monophosphate level in the liver. Vagal stimulation also had no effect on the level. 4. The activity of phosphorylase phosphatase decreased promptly (within 30 s) and markedly on splanchnic-nerve stimulation, but did not change significantly on administration of epinephrine of glucagon. A small but insignificant increase in phosphatase activity wasobserved upon vagal stimulation. 5. The effect of Ca-2+ on purified dephospho-phosphorylase kinase was studied. The activity was found to depend partially on free Ca-2+ at low Ca-2+ concentrations (1-10-minus 7--1-10-minus 5 M). 6. These results suggest that the rise in hepatic phosphorylase content upon splanchnic-nerve stimulation, unlike that induced by epinephrine and glucagon, is not mediated by adenosine 3', 5'-monophosphate and subsequent activation of dephospho-phosphorylase kinase, but rather by inactivation of phosphorylase phosphatase. The possible existence of a new factor in this mechanism is discussed.     

Characterization of Newly Formed and Aged Granules in the Neurohypophysis

Neurosecretory granules from the rat and bovine neurohypophysis were isolated and some of their biochemical and biophysical properties studied. Neurosecretory granules (NSG) from rat neurohypophysis were labeled, in vivo, with [35S]cysteine and isolated on isoosmotic gradients. Whereas 1 day after labeling most of the radioactivity was found in the lower part of the gradient, 35 days later the isotope was also located in the lighter NSG-containing fraction. Different analytical procedures showed that the lighter fraction, both in bovine and rat NSG, contain more subpopulations of neurophysin-like material than the heavier fraction. The first material to be released during stimulation of secretion, in vivo or in vitro, is mobilized from the heavy NSG. Isolation of rat NSG, at different times during and after dehydration of the animals, reveals that the newly synthesized material is found in the heavy NSG-containing fraction. Furthermore, the results indicate that the newly synthesized NSG are more resistant to lysis than the lighter granules. The results are discussed in relation to the maturation and degradation processes of the granule content and to the functional state of the NSG.     

乙酰胆碱和A23187对离体大鼠肾上腺髓质细胞肾上腺素分泌的作用

通过胆碱能激动剂乙酰胆碱及离子诱导剂A23187（以下简称激动剂）作用于分离的肾上腺髓质细胞,以引起离体细胞的刺激－分泌耦联过程,运用细胞立体形态计量法计算分泌过程中的嗜铬颗粒数目的变化,运用电镜X射线显微分析法测量分泌过程中嗜铬颗粒内钙含量的变化,并运用高 液相色谱分析法测定离体细胞在激动剂作用后的肾上腺素分泌情况,结果发现,分离的肾上腺髓质细胞嗜铬颗粒内钙含量在激动剂作用10min时有明显下降,颗粒数目在激动剂作用过程中呈缓慢下降趋势,而细胞悬液中的肾上腺素含量在激动剂作用20min以后有明显的升高,激动剂作用引起的离体肾上腺髓质的细胞分泌时颗粒内钙含量的下降早于颗粒数目减少或肾上腺素升高,提示颗粒释放的Ca^2 可能是引起细胞分泌的原因之一。     

Epinephrine intracerebroventricular stimulation modifies the LH effect on ovarian progesterone and androstenedione release

This study investigates the interaction between the effect of epinephrine intracerebroventricular (icv) injection and LH on the progesterone concentration in ovarian vein blood (Po) in vivo, and also, on the release of ovarian progesterone and androstenedione in vitro, in rats on dioestrus day 2. When 2 mg ovine LH were injected in vein (i.v.), Po increased reaching 120+/-12.2 and 151+/-17.5 ng ml(-1) at 22 and 25 min, respectively. Another group of rats was injected intracerebroventricular with 5 microgram epinephrine at time zero, and with 2 mg ovine LH i.v. 3 min later. This time Po decreased during the first 3 min, then increased, reaching 64+/-7.1 ng ml(-1) at 25 min, lower than the Po obtained 22 min after LH i.v. injection only (P<0.01). Moreover, rats were injected i.v. with 2 mg ovine LH at time zero, and 7 min later with epinephrine intracerebroventricular. Po increased during the first 7 min, decreased until the 13th minute and reached 70+/-8.9 ng ml(-1) at 25 min, lower than the Po obtained 25 min after LH i.v. injection only (P<0.01). In other experience, rats with one (either right or left) superior ovarian nerve transected (SON-t), were injected intracerebroventricular with epinephrine. Five minutes later, the ovaries were removed and incubated in vitro with LH. Both ovaries (right or left) in which the SON was intact at time of epinephrine i. c.v. injection, showed a reduction of progesterone and androstenedione released in vitro (P<0.05). These results suggest that, on dioestrus day 2, the central adrenergic stimulus competes with LH in the release of ovarian progesterone. Also, the neural input that arrives at the ovary through the SON would antagonize the ovarian progesterone and androstenedione response to LH.     

Membrane routing during exocytosis and endocytosis in neuroendocrine neurones and endocrine cells: use of colloidal gold particles and immunocytochemical discrimination of membrane compartments

Summary The hypothesis that the retrieval of membranes of neurohypophysial neurosecretory granules (NSG) and small electron-lucent microvesicles occurs by different routes was tested by incubating neurohypophysial neurosecretosomes with colloidal gold particles of various sizes. Neurosecretosomes derived from normal Long Evans rats and incubated in media of normal ionic composition endocytosed a few small (<25 nm) gold particles into 40–50 nm electron-lucent microvesicles. After depolarisation, more small gold particles were found in microvesicles, and small and large (>25 nm) gold particles in vacuoles. Oxytocin-containing neurosecretosomes derived from Brattleboro rats, which contain 160 nm-diameter NSG, endocytosed gold particles in a pattern indistinguishable from that of neurosecretosomes from Long Evans rats. However, neurosecretosomes derived from defective vasopressin neurones of Brattleboro rats, which contain microvesicles, small vacuoles, and a few 100 nm dense-cored vesicles, but not 160 nm NSG, endocytosed only small colloidal gold particles. Early after depolarisation the gold particles were present only in microvesicles, but later some could be found in vacuoles and lysosome-like structures. Immunogold cytochemistry using a polyclonal antiserum raised against microvesicle-rich neurosecretosomes derived from Brattleboro rats labelled microvesicles in the posterior pituitary strongly, NSG weakly, and vacuoles to a variable extent. These data together indicate that, after exocytosis, the membranes of NSG are recaptured as large vacuoles. Microvesicles are exocytosed and endocytosed separately.     

Studies on the mechanisms of epinephrine stimulation of lypolysis

The time course for epinephrine stimulation of lypolysis, cyclic AMP accumulation and activation of protein kinase was studied in adipose tissue from hypophysectomized rats. Triglyceride breakdown, as assessed by glycerol release, increased rapidly in response to epinephrine, maintained a constant rate as long as the hormone was present, and decreased rapidly to basal values when the hormone was removed. Cyclic AMP accumulation was transient peaking within 3 min of exposure to epinephrine and then declining to levels indistinguishable from basal by 9 min. Protein kinase activity in extracts also peaked at 3 min and thereafter declined to a level approximately 25% greater than resting activity. Peak levels of cyclic AMP, steady state levels of protein kinase activity and the rate of glycerol production were all related in a dose dependent manner to the concentration of epinephrine. These observations suggest that the spike in cyclic AMP levels may be necessary to trigger the activation of lipolysis, but was not sufficient to sustain an accelerated rate of tryglyceride breakdown. Continued activation of protein kinase, however, may be essential to sustained lipolysis.     

Studies on the mechanisms of epinephrine stimulation of lypolysis.

The time course for epinephrine stimulation of lypolysis, cyclic AMP accumulation and activation of protein kinase was studied in adipose tissue from hypophysectomized rats. Triglyceride breakdown, as assessed by glycerol release, increased rapidly in response to epinephrine, maintained a constant rate as long as the hormone was present, and decreased rapidly to basal values when the hormone was removed. Cyclic AMP accumulation was transient peaking within 3 min of exposure to epinephrine and then declining to levels indistinguishable from basal by 9 min. Protein kinase activity in extracts also peaked at 3 min and thereafter declined to a level approximately 25% greater than resting activity. Peak levels of cyclic AMP, steady state levels of protein kinase activity and the rate of glycerol production were all related in a dose dependent manner to the concentration of epinephrine. These observations suggest that the spike in cyclic AMP levels may be necessary to trigger the activation of lipolysis, but was not sufficient to sustain an accelerated rate of triglyceride breakdown. Continued activation of protein kinase, however, may be essential to sustained lipolysis.     

Correlation between suckling-induced changes in the ultrastructure of mammotrophs and prolactin release

Effects of suckling on the structure of mammotrophs and the release of prolactin, were studied in rats on the 10th day of lactation with the use of electron microscopy and radioimmunoassay techniques. Nursing animals were separated from their young for 8 hr and subsequently united and permitted to nurse for 1, 5, 15, 30 min; or 1, 2 and 4 hr. Blood samples were obtained prior to and throughout the suckling interval and pituitary glands were processed for electron microscopy. Control animals consisted of normal lactating females and animals separated from their young for 8 hr. Normally lactating controls had high prolactin serum levels (501 +/- 95 ng/ml) and synthetically active appearing mammotrophs. An 8 hr separation from the pups induced a dramatic lowering of serum prolactin (32 +/- 5 ng/ml), an increase in secretory granule storage, and a great dilation of rough endoplasmic reticulum (RER) cisternae. Five min of renewed suckling resulted in a rise of plasma prolactin levels (605 +/- 183 ng/ml) which remained high thereafter. The major ultrastructural changes observed during the first 30 min of suckling were as follows: 1) at 1 min, the RER became cmone?); 2) AT 5 MIN, AND MUCH MORE OBVIOUSLY AT 15 AND 30 MIn, a massive discharge of secretory granules was observed; and 3) at 15 min, the collapsed RER underwent transformation for 1,2 and 4 hr) induced new hormone synthesis as suggested by the presence of hypertrophied Golgi elements and numerous immature granules. This was accompanied by a new transformation of the RER from the vesicular into a lamellar form now consisting of very slender cisternae lined with numerous ribosomes, presumably involved in the renewal of the synthetic process. The morphologic findings described correlate well with the time table of prolactin release. In addition, the dramatic early changes in the structure of the RER suggest a possible involvement of this organelle in the storage and release of a proposed rapidly releasable pool of prolactin.     

Dynamics of secretory granules in somatotrophs of rats after stimulation with growth hormone-releasing factor: a stereological analysis

The anterior pituitary tissue of male rats injected with growth hormone-releasing factor (GRF) was either processed for stereology at the light-and electron-microscopic levels, or homogenized for growth hormone (GH) assay 2–60 min after GRF injection. Secretory granules of somatotrophs became smaller but increased in numerical density 2 min after GRF injection. Their volume density began to increase at 5 min. The frequency of exocytosis of the granules was most prominent as early as 2 min after GRF injection and reduced thereafter. GH levels in the tissue were lowest at 2–5 min, and returned to the control value by 60 min. Serum GH levels were highest at 15 min; even at 60 min, this value was higher than in the controls. These findings suggest that secretory granules in somatotrophs are stimulated to divide by GRF, resulting in a decrease in size and an increase in number. The discrepancy between the earlier formation of new secretory granules and the later restoration of intracellular GH levels implies that GRF first stimulates the synthesis of constituents of granules other than GH, and only later the synthesis of GH, and that newly formed small secretory granules contain less GH. From the clearance rate of serum GH and the frequency of granule exocytosis, it can be estimated that about a half million granules are released to maintain 1 ng/ml of serum GH in rats.     

Epinephrine is unessential for stimulation of liver glycogenolysis during exercise

To determine the role of adrenal medullary hormones in controlling the rate of liver glycogenolysis during exercise, adrenodemedullated (ADM) and sham-operated (SO) rats were run on a rodent treadmill at 21 m/min up a 15% grade for 0, 30, or 60 min. Rats were anesthetized by intravenous injection of pentobarbital sodium, and liver, muscle, and blood were collected and frozen. Liver glycogen decreased at similar rates in ADM and SO rats. Hepatic adenosine 3',5'-cyclic monophosphate (cAMP), plasma glucagon, and plasma free fatty acids increased to the same extent in both ADM and SO rats. The adrenodemedullation caused a reduction in glycogenolysis in the fast-twitch white region of the quadriceps, soleus, and lateral gastrocnemius during exercise. The normal exercise-induced increase in blood glucose and lactate and the decline in plasma insulin were not observed in the demedullated rats. During submaximal exercise the principal targets for epinephrine released from the adrenal medulla appear to be pancreatic beta-cells and skeletal muscle and not the liver.     

The hypothalamic magnocellular neurosecretory system of the guinea pig. III. Ultrastructure of the fetal neural lobe.

The ultrastructure of the fetal guinea pig neural lobe was studied from day 40 to day 60 of gestation. Pituitaries were taken every five days and at least four glands from each gestational period were examined. Bundles of nerve fibers had invaded the periphery of the gland on day 40. By day 50 axon profiles were distributed throughout the entire posterior pituitary though pituicyte processes continued to act as a barrier between axons and the perivascular space of capillaries. Neural processes established contact with the capillaries between days 55 and 60. Neurosecretory granules (NSG) were present within a few axons on day 40. The number of axons with NSG and the total quantity of granules increased gradually throughout fetal development. Electron-lucent granules (microvesicles) were observed infrequently until the day of birth. A population of dense-cored vesicles, 70-80 mmu in diameter, was present from day 50 onward; a second population with larger diameters was also present throughout the developmental sequence and these increased from 90-130 mmu in diameter to 170-220 mmu in diameter between days 40 to 60. The presence of neurosecretory granules is discussed in relation to the onset of synthesis and storage of neurohypophysial hormones.     

The effects of adrenalectomy on the alpha-adrenergic regulation of cytosolic free calcium in hepatocytes

We have previously published that bilateral adrenalectomy in the rat reduces the Ca2+-mediated alpha-adrenergic activation of hepatic glycogenolysis, while it increases the cellular calcium content of hepatocytes. In the experiments presented here, the concentration of cytosolic free calcium (Ca2+i) at rest and in response to epinephrine was measured in aequorin-loaded hepatocytes isolated from sham and adrenalectomized male rats. We found that in adrenalectomized rats the resting Ca2+i was elevated, the rise in Ca2+i evoked by epinephrine was reduced, and the rise in 45Ca efflux that follows such stimulation was depressed. Furthermore, the slope of the relationship between Ca2+i and calcium efflux was decreased 60% in adrenalectomized. Adrenalectomy did not change Ca2+ release from intracellular calcium pools in response to IP3 in saponin-permeabilized hepatocytes. The EC50 for inositol 1,4,5-triphosphate and the maximal Ca2+ released were similar in both sham and adrenalectomized animals. Finally, the liver calmodulin content determined by radioimmunoassay was not significantly different between sham and adrenalectomized rats. These results suggest that 1) adrenalectomy reduces calcium efflux from the hepatocyte, probably by an effect on the plasma membrane (Ca2+-Mg2+)-ATPase-dependent Ca2+ pump and thus alters cellular calcium homeostasis; 2) adrenalectomy decreases the rise in Ca2+i in response to epinephrine; 3) this decreased rise in Ca2+i is not due to defects in the intracellular Ca2+ storage and mobilization processes; and 4) the effects of adrenalectomy on cellular calcium metabolism and on alpha-adrenergic activation of glycogenolysis are not caused by a reduction in soluble calmodulin.     

Effects of adrenergic agonists and antagonists on muscle O2 uptake and lactate metabolism

To investigate adrenergic receptor-mediated responses in dog gastrocnemius-plantaris muscle, several catecholamine agonists, isoproterenol, epinephrine, norepinephrine, and phenylephrine, and two antagonists, propranolol and phenoxybenzamine, were given during repetitive, isotonic, tetanic contractions. The response variables that were measured were muscle blood flow, shortening during constant load contractions, and arterial and venous O2 and lactate concentrations. The calculated variables were O2 uptake (VO2), net lactic acid output (L), and power output. In the control experiments, the contractions increased VO2 to approximately 50 times rest by 2 min. Thereafter, shortening, work, and VO2 declined together by 17% at 30 min, indicating muscle fatigue. L increased rapidly to nearly 0.8 mumol X g-1 X min-1 by 2 min, declined to 0.3-0.4 mumol X g-1 X min-1 by 7 min, and was like rest at 15, 22.5, and 30 min. The arterial lactate concentration rose steadily from rest to 30 min of contractions. Epinephrine infusion stopped the decline of VO2 during the contractions, but this effect was not observed with the other agonists. Propranolol decreased VO2 compared with controls at 22.5 and 30 min of contractions. Phenoxybenzamine decreased VO2 compared with controls at all times during contraction, and the decline with time was present. Coinfusion of epinephrine with propranolol reduced the decline in VO2 observed with propranolol alone. Both epinephrine and isoproterenol increased L compared with controls. This epinephrine response was antagonized by propranolol but enhanced by phenoxybenzamine. Both isoproterenol and epinephrine infusions increased arterial lactate concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pituitary cyclic AMP and plasma hormone responses to epinephrine administration in vivo

The present study was conducted to characterize the in vivo effects of epinephrine administration on levels of pituitary cyclic AMP and plasma hormones. Rats were injected with saline or epinephrine bitartrate (1 mg/kg lP) and sacrificed by decapitation 1, 5, 15, 30 or 60 min post-injection. Levels of pituitary cyclic AMP and plasma ACTH, beta-endorphin, beta-LPH, corticosterone and prolactin were determined by radioimmunoassays. The injection procedure itself was somewhat stressful as demonstrated by increased levels of plasma prolactin and ACTH 5 min following either saline or epinephrine injection. This "stress" response was rapid and short-lasting for the pituitary hormones. The response of the adrenal hormone, corticosterone, to saline injection was slower in onset and longer in duration. Pituitary cyclic AMP levels did not increase following saline injection. Epinephrine-injected animals displayed markedly elevated plasma levels of ACTH, beta-endorphin and beta-LPH at 15, 30 and 60 min as compared to control or saline-injected rats. In addition, levels of pituitary cyclic AMP were increased over 10 fold at these times. Levels of plasma prolactin, a stress-responsive hormone, were not significantly increased in epinephrine-injected animals as compared to saline-injected rats indicating that these later responses seem to be specific to epinephrine rather than to stress.     

Factors Affecting the Germination of Septoria nodorum Pycnidiospores

Most pycnidiospores of Septoria nodorum were released from pycnidia into water in 30 min, more than 50% being released in 10 min. The viability of pycnidiospores in aqueous suspension, assessed by germination on a selective agar medium, decreased more rapidly in daylight than in darkness but no spores germinated after 50 h. The presence of cirrhus extract stimulated germination of pycnidiospores in suspension.     

Ultrastructure of neurosecretory cells in the frontal ganglion of the tobacco hornworm, Manduca sexta(L)

Ultrastructural evidence is presented to indicate the synthesis, storage, and packaging of neurosecretory material in the frontal ganglion of Mandaca sera. Two morphologically distinct stages in the development of NSG were observed in the NSC. The immature granule contains granular electron dense material and arises from dilations of the rough endoplasmic reticulum. In diapause pupae the immature granules accumulate, whereas in developing larvae and pupae, they fuse with the proximal face of the Golgi apparatus. The mature granule contains opaque electron dense material and is seen in close association with the distal cisternae of the Golgi apparatus in developing pupae and larvae. The mature granules presumably contain the material from the immature granules which has been chemically altered and condensed by the Golgi apparatus and probably represent the mature NSG.The second process in neurosecretory cells of developing larvae is cyclic and the various stages are described. A comparison of neurosecretion in photoperiodically induced diapause and developing (non-diapause) larvae and pupae is presented.