Differences between adrenomedullary adrenaline and noradrenaline cells: quantitative electron-microscopic evaluation of their differential cellular association with supporting cells.

Quantitative differences in cellular association of adrenomedullary chromaffin cells with other types of cells, mainly supporting cells, were studied. Adrenaline (A) and noradrenaline (NA) cells were compared. Electron micrographs (12000 x) of profiles of A and NA cells, bordering against other types of cells, were used for quantitative evaluation. Supporting cells constituted the majority of the non-chromaffin cell types. Occurrence frequencies of chromaffin cells contiguous with other types of cells were: (1) higher for A cells (68.9%, 199/289) than for NA cells (11.0%, 34/309) in case of small contact regions (chi 2-test: P < 0.001), and (2) higher for NA cells (68.3%, 211/309) than for A cells (9.7%, 28/289) in case of extended contact regions (P < 0.001). In conclusion, the extent of cellular association with supporting cells was remarkably lower in A cells than in NA cells. Such an arrangement is likely to be appropriate for the extensive, homogeneous control and amplified response characteristic of A cells, and for the close range, complex control and more diverse responses characteristic of NA cells.

Rough endoplasmic reticulum in the adrenaline and noradrenaline cells of the adrenal medulla: effects of intracranial surgery and pinealectomy.

UNLABELLED: Adrenal medullas in 53-day-old rats of the nonoperated (NO) group (n = 31), the sham-operated (SPX) group (n = 35) and the pinealectomized (PX) group (n = 38) were examined electron microscopically 14 days after surgery. Cell profiles showing solitarily and sparsely distributed rough endoplasmic reticulum (RER) were most frequent in the PX group (daily mean: 66.9%, 427/638), less in the NO (56.0%, 336/600), and least in the SPX (48.5%, 297/612) in adrenaline (A) cells (chi 2-test: P less than 0.001), while most frequent in the NO group (68.8%, 340/494), less in the PX (64.3%, 303/471), and least in the SPX (57.4%, 256/446) (P less than 0.005) in noradrenaline (N) cells. Individual variation was less in A cells than in N cells. Cell profiles showing a large accumulation of RER was more frequent in A cells (NO:8.5%, SPX:13.1%, PX:7.7%) than in N cells (NO:2.8%, SPX:4.5%, PX:4.7%) (controls: P less than 0.001). Sham pinealectomy increased a large accumulation of RER in A cells (P less than 0.02) and a small aggregation of RER in N cells (P less than 0.005) with opposite effects of pinealectomy (P less than 0.005, P less than 0.025). Pinealectomy decreased a small aggregation of RER without effects of sham pinealectomy in A cells (P less than 0.001). CONCLUSIONS: (1) Accumulation or aggregation of RER in adrenomedullary chromaffin cells was influenced from the pineal gland either as or without effects of intracranial surgery, and (2) RER in adrenomedullary chromaffin cells showed differences due to cell types.

Effects of pinealectomy on the mitotic activity of adrenomedullary chromaffin cells in relation to time of day.

The frequency of mitoses of adrenaline (A) cells and noradrenaline (N) cells in the adrenal medulla of nonoperated (NO), sham-operated (SPX), and pinealectomized (PX) male, 53-day-old Holtzman rats (n = 133) was investigated by means of light microscopy. Animals were killed at eight time points during a standardized 24-h light-dark (12:12) cycle 14 days after surgery. Mitotic indices (n/1,000) were determined in sections of adrenal medulla fixed with glutaraldehyde and OsO4. Overall frequency of mitoses was extremely low (mitotic index: 0.73 = 115/157,223). Daily mean mitotic index was maximum in A cells (0.83) and minimum in N cells (0.52) of PX group but did not show statistically significant differences between cell types or experimental groups. Neither cell type in NO animals showed 24-h changes in mitotic index, but cells in SPX animals did, with highest value in the late dark phase and lowest in the late light phase, when values of two cell types were combined (P less than 0.01-0.001). In PX animals, mitotic index followed a similar but more distinct 24-h change in A cells (P less than 0.009), but not in N cells, resulting in different time-of-day changes between two types of cells (P less than 0.01-0.05). The mitotic index was higher in PX than in control (NO and SPX) animals in the middark phase (P less than 0.05) and lower in operated (SPX and PX) than in nonoperated (NO) animals from late light to the early dark phase, suggesting that the latter was possibly due to a residual effect of the surgery. These results are consistent with the interpretation that the pineal has an inhibitory action on A cells and may coordinate the two types of cells in their mitotic activity, especially in the middark phase of the daily cycle.

Changes with darkness in regional brain 5-hydroxytryptamine and 5-hydroxyindole acetic acid: local differences with pinealectomy, sham surgery, and melatonin.

Brain regional 5-HT (5-hydroxytryptamine) and 5-HIAA (5-hydroxyindole-3-acetic acid) concentrations in male LE rats at three times were measured by a fluorescence method, to evaluate effects of intracranial surgery and administration of melatonin on the changes in these compounds during the first part of the dark phase of the daily cycle in a fixed 12:12 L:D photoperiod. Early surgical pinealectomy or a similar but sham intracranial surgery, led to delay in darkness-associated fall in frontal cortical and striatal 5-HT. A single melatonin injection two hours before darkness, reversed this effect in frontal cortex but not striatum. Melatonin's specification of action in this experiment is interpreted as residing in specific timing of release and/or administration, rather than in either a fixed or general basis neurochemically.

Quantitative cytological analysis of functional changes in adrenomedullary chromaffin cells in normal, sham-operated, and pinealectomized rats in relation to time of day: III. Nuclear density.

The sizes of adrenaline (A) and noradrenaline (N) cells in the adrenal medulla of nonoperated (NO), sham-operated (SPX), and pinealectomized (PX) male rats (n = 126) were investigated by quantitative light microscopy. Animals were killed at eight time points during a standardized 24-h, light-dark (12:12) cycle 14 days after surgery. Nuclear densities were measured in semithin sections of epon-embedded specimens, initially fixed with glutaraldehyde and OsO4. Major findings are as follows. 1) The mean size of adrenomedullary A cells throughout 24 h (P less than 0.001), especially in the dark phase (P less than 0.001) but not in the light phase, was larger in PX animals than in NO and SPX animals. There were no statistically significant differences in the size of N cells among the three experimental groups in either the dark phase or the light phase. 2) The sizes of A and N cells showed time-of-day changes in the NO and the SPX animals but not in the PX animals. The temporal relationship of 24-h changes in the cell size tended to be different between A and N cells in the NO and the SPX animals but not in the PX animals. 3) The cell size was apparently larger in A cells than in N cells in each experimental group. Pinealectomy thus caused hypertrophy of A cells, especially in the dark phase, but not apparently hypertrophy of N cells. Concerning the pinealectomy effects in relation to the time of day, the results support the hypothesis of pineal action being phase-tuning and coordinating of at least some circadian systems.

Effects of thyroid hormone on light/dark melatonin synthesis and release by young and maturing rat pineal glands in vitro.

Synthesis and release of melatonin were studied in pineal explants from 14- (young) and 60-day-old ('maturing') male Long-Evans rats with or without added thyroid hormone, triiodothyronine (T3), at or near physiological levels and under light and dark conditions. Incubation for 6 hr (1200-1800) was in a synthetic medium; melatonin was measured by radioimmunoassay (RIA). In light, T3 increased melatonin levels in pineal and medium of cultures from either young or maturing animals. In dark, T3 decreased melatonin levels in the pineals of either age, but was without significant effect on levels in the medium. Since it is known from other work that 14-day-old rat pineal glands do not yet have a complete sympathetic innervation system, it is here doubly evident that T3 can modulate directly pineal synthesis and release of melatonin, and may not depend upon a mature sympathetic innervation. Light in the studied conditions was permissive from the stimulatory action of T3 on pineal synthesis and release of melatonin in vitro.

Quantitative cytological analysis of functional changes in adrenomedullary chromaffin cells in normal, sham-operated, and pinealectomized rats in relation to time-of-day: II. Nuclear-cytoplasmic ratio, nuclear size, and pars granulosa of nucleolus.

Adrenaline(A)- and noradrenaline(N)-cells in the adrenal medulla of nonoperated (NO), sham-operated (SO), and pinealectomized (PX) male rats (n = 125) were investigated by quantitative electron and light microscopy. Animals were killed at eight time points during a standardized 24-h, light-dark (12:12) cycle 14 days after surgery. Nuclear-cytoplasmic (N/P) ratios, diameters of nuclei, and the frequency of nucleoli showing a large amount of pars granulosa (granulated nucleoli), were the primary characteristics studied. Major findings include the following: 1) The frequency of low N/P ratios over a 24-h period tended to be higher in PX animals than in controls in A-cells, as shown in large cell profiles (P less than 0.02); but such a tendency was not apparent in N-cells. Daily mean nuclear diameters were similar among the three experimental groups. 2) The 24-h changing pattern of phase relations in the frequency of low N/P ratio and nuclear size differed between A- and N-cells in NO and SO but not in PX animals. 3) The frequency of granulated nucleoli in A-cells was much higher in PX animals than in NO and SO animals throughout a 24-h period (P less than 0.018), especially from the late light to early dark phase (P less than 0.003), and higher in A-cells than in N-cells generally (P less than 0.0009). Pinealectomy thus caused increases in the cytoplasm and in the pars granulosa of the nucleolus in many A-cells; changes in N-cells were less apparent. This suggest a disturbed balance and coordination between A- and N-cell systems of adrenal medulla following pinealectomy.

Developmental changes in hypothalamic melatonin levels of male rats.

Hypothalamic melatonin levels of Long-Evans male rats were studied at three ages (25, 55-60 and 90 days), at four times of the day in the autumn (6:00, 12:00, 18:00 and 24:00), and at two times (12:00 and 24:00) in the spring using radiommunoassay. Melatonin levels increased markedly at noon at 55-60 days of age, compared with the levels at the same time of the day at 25 and 90 days. This increase persisted in autumn and spring. The 24-hr pattern in hypothalamic melatonin was the inverse of that in the pineal, with the levels at noon higher than those at midnight. This pattern was detectable at 25 days of age although the difference in melatonin between 12:00 and 24:00 hr was not great. The day/night difference was prominent by 55-60 days of age and disappeared by adulthood (90 days). This 24-hr pattern was similar in spring and autumn in the three ages studied. Although in the 55-60-day-old group the melatonin ratio (noon/midnight) was the same in autumn and spring, the absolute levels of melatonin in spring were significantly lower. The findings are consistent with the general concept of a modulatory role of melatonin in control of hypothalamo-hypophyseal GnRH and gonadotropin function, and the timing of the developmental maturation of this neuroendocrine axis. Demonstration of the mechanism of melatonin's action at the hypothalamic level will be facilitated by further definition of quantitative developmental changes.

Spontaneous continuous release of spermatozoa and its predawn surge in male passerine birds.

Spontaneous, continuous release of morphologically normal spermatozoa occurs in males of species of passerine (order Passeriformes) birds that were examined. It was demonstrated and studied quantitatively in temporarily captive and isolated house sparrows and house finches by means of repetitive cloacal lavages and extraction of excreta. It is suggested that this phenomenon could be exploited to facilitate comparative and quantitative evaluations of release of spermatozoa in relation to diverse environmental, physiological, and social factors.

Lower tryptophan:phenylalanine ratios in culture media increase medium: pineal melatonin ratios in early dark but not late light phase.

Pineals from male Long-Evans rats (60-65 days old; adapted to a 0700-1900 photoperiod) were cultured for 6 h either in light (1200-1800) or in dark (1800-2400). The objective was to ascertain the effects of tryptophan (trp) and phenylalanine (phe) levels and ratios in the culture medium on melatonin levels in the pineals and their respective media. Total culture (pineal + medium) melatonin levels, determined by RIA, were similar under all conditions. However, in cultures during the early dark phase (1800-2400) lower trp:phe ratios in the medium led to lower pineal:medium ratios of melatonin content. In cultures during the late light phase (1200-1800) the trp:phe ratio had little impact on the pineal:medium distribution of melatonin. Trp:phe ratio rather than absolute level of either amino acid appeared responsible for this effect. Functionally this means that during early dark phase, but not late light phase, movement of melatonin from cultured pineal to medium is progressively facilitated by lower trp:phe ratios. It remains to be determined to what extent darkness per se and/or endogenous pineal rhythmic mechanisms have a permissive role in the action of trp:phe ratio on pineal melatonin release. A melatonin compartmentalization/release effect of these or other amino acids, or their ratios, has not been reported previously and may possibly contribute to mechanisms for melatonin's transport or release at night.

Effects of melatonin implants on structures and behaviors of the house finch (Carpodacus mexicanus) eye.

This study aimed to determine the extraretinal effects of melatonin upon the eyes of an avian species, the House Finch (Carpodacus mexicanus). Twelve birds (full-grown, second-year males) each received a Silastic tubing intraperitoneal implant, six containing melatonin (average release = 24 micrograms/d/bird; = M birds) and six being empty (= C birds). Microscopic study of pupillary and palpebral behaviors during the final week demonstrated lesser pupillary diameters and interpalpebral distances in M birds under all test conditions. These effects could have diminished mean light levels reaching parts of the retina. Characteristics of the relative miosis and ptosis of M birds resemble signs in some CNS disorders, such as altered inhibition of the Edinger-Westphal nucleus, and especially lesions in, or lowered activity of, higher sympathetic centers (a subtype of Horner's syndrome). Weights of eyes and their parts were the same in M and C birds, contrasting with previously reported results from male Golden Hamsters, possibly due to species differences and/or preexperimental attainment of full growth in the finches. Effects of melatonin on pupillary and palpebral behaviors, demonstrated here for the first time, foster caveats for simplistic experimental designs and interpretations with melatonin when sensory-neural-behavioral interactions are affected. Quantitative changes in pupillary and palpebral behaviors may, nevertheless, provide a window for monitoring central actions of melatonin in living test subjects in chronic studies.

Effects of melatonin on adrenomedullary dopamine-beta-hydroxylase activity in golden hamsters: evidence for pineal and dose dependencies.

Pineal influence in the control of adrenomedullary function in golden hamsters was investigated by examining changes in adrenal dopamine-beta-hydroxylase (DBH) activity following pinealectomy, either alone or in combination with melatonin administration. Adult males acclimated to an LD 14:10 photoperiod were distributed in five experimental groups: intact controls (NO), sham-pinealectomized (S), sham-pinealectomized with black plastic shielding of the pineal region, pinealectomized (PX), and pinealectomized with the operated region shielded. Animals representing all of these groups were injected (between L11 and L11.75) with either vehicle, or a low dose (25 micrograms) or a high dose (2,500 micrograms) of melatonin daily for 28 days, after which they were killed, and the adrenals were collected for assay of DBH activity by means of a sensitive radioenzymatic method. We found that PX + vehicle led to increased (P less than .05) adrenal DBH activity in comparison with either NO or S groups; daily 25 micrograms of melatonin resulted in lowered DBH activity in the NO group when compared with NO + vehicle (P less than .001) or S + vehicle (P less than .001) groups; PX + 25 micrograms melatonin reversed the action of 25 micrograms melatonin in the NO + 25 micrograms group; 2,500 micrograms melatonin was without effect on adrenal DBH in any of the injected surgical groups. These results show an inhibitory pineal influence on adrenal DBH activity, and that this was dose dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Double isotopic method using dansyl chloride for the determination of GABA in rat C6 astrocytoma cell cultures.

Methods are described for the quantitative measurement of GABA in culture. The method can be adapted to any amino acid or dansyl-chloride-reactive species. The sensitivity and selectivity of the procedure result from the double isotopic design in which (14C)-labeled internal standard was added to the samples before reaction with (3M)-labeled dansyl chloride. Values obtained by ion-exchange amino acid analysis of cultures agree closely with the values obtained by the double isotopic method. This method is sensitive enough to measure GABA intracellularly and the condition medium.

Quantitative ultrastructural analysis of differences in exocytosis number in adrenomedullary adrenaline cells of golden hamsters related to time of day, pinealectomy, and intracellular region.

This research analyzed differences mainly in the incidence of exocytotic figures in adrenaline cells (A-cells) in pinealectomized (PX), sham-operated (SPX), and non-operated (NO) adult male golden hamsters, with the aim of determining whether these parameters change with the time of day and following pinealectomy, and whether intracellular regional differences exist in such changes. Animals acclimated to a standardized light:dark (LD) 12:12 photoperiod were sacrificed at 11 h after the onset of light (L-11h) and 1 h after the onset of darkness (D-1h) (8 animals/group/time) at 28 days postoperation. The adrenal medullas were examined and analyzed morphometrically by electron microscopy. The number of exocytoses per unit length (NEL) and the exocytosis index (a rough index of the number of exocytoses per cell) were measured in PF (perivascular-space-facing) and non-PF plasma membranes. NEL increased from L-11h (NO: 0.040 +/- 0.010, mean +/- SE) to D-1h (0.078 +/- 0.012) in all three experimental groups (ANOVA: P less than 0.005), showing over fourfold higher levels in PF than in non-PF membranes. NEL in PF membranes in PX animals showed higher levels than those in NO and SPX animals (P less than 0.025), but in non-PF membranes, no differences owing to time of day or surgery were seen. Exocytosis indices were (1) higher at D-1h than at L-11h in all three experimental groups (P less than 0.005), (2) similar in PF and non-PF membranes in control groups, and (3) higher in PF membranes in the PX group than in either non-PF membranes or PF membranes in control groups. In conclusion, the exocytosis number in A cells changes in relation to time of day, rising in early dark phase, and its rise following pinealectomy can be seen only in PF membranes.

Increases in volume, fluid content, and lens weight of eyes following systemic administration of melatonin.

Melatonin's effects were studied in male golden hamsters (Mesocricetus auratus) distributed among five surgical groups (nonoperated, sham-pinealectomized, sham-pinealectomized plus black plastic shielding of the pineal region, pinealectomized, and pinealectomized plus black plastic shielding of the pineal region) and three injection groups (vehicle only, 25 micrograms melatonin, and 2,500 micrograms melatonin). Injections (s.c.) were daily for 28 d at L11 to L11.75 in a (light:dark) L:D 14:10 artificial photoperiod. Animals (N = 112) were killed and dissected on the day after the last injection (at 55-65 d of age). None of the surgical procedures affected weights of eyes or their parts, nor did they influence the effects of administered melatonin on the eyes. Melatonin caused an increase in absolute and relative eye weight and an increase in fluid content of intraocular space. The magnitudes of these effects were positively related to melatonin dose. These same eyes had a progressively lower weight of nonlenticular tissues with low to high doses of melatonin, probably in relation to greater fluid content, and suspected increase in intraocular pressure. Lens wet and dry weights were significantly greater in animals receiving melatonin, but only at the high dose. These actions of melatonin are likely to be direct and are shown to not require the presence of the pineal. Experiments of other designs are suggested in order to determine whether the effects of the low, near physiological, dose of melatonin represent physiological actions of endogenous melatonin, synthesized and released within the eye. However, effects of large doses of melatonin on the eye are still noteworthy in relation to interpretation of experiments employing such dosages, and of disease states involving changes in intraocular pressure.

Quantitative cytological analysis of functional changes in adrenomedullary chromaffin cells in normal, sham-operated, and pinealectomized rats in relation to time of day: I. Nucleolar size.

Adrenaline (A)- and noradrenaline (N)-cells in juxtacortical and central areas of adrenal medullas of nonoperated, sham-operated, and pinealectomized male rats (n = 125) were investigated by quantitative electron and light microscopy. Animals were killed at eight time points during a standardized 24-h, light-dark (12:12) cycle 14 d after surgery. Diameters of nucleoli (n = 9,600) and the nucleolar margination rate were studied chiefly by light microscopy. Major findings include the following: Twenty-four-hour changes occurred in nucleolar diameters in adrenomedullary chromaffin cells in nonoperated animals, with the peak in the late dark phase (X +/- SD, A-cells: 1.29 +/- 0.06 micron; N-cells; 1.23 +/- 0.05 micron) and the minimum in the mid-light (A-cells: 1.16 +/- 0.04 micron) or early dark (N-cells: 1.14 +/- 0.03 micron) phase. These changes were markedly dampened (diminished) in sham-operated animals. Since this dampening of time-of-day changes after sham-operations was not seen in A-cells after pinealectomy, mediation by the gland of this dampening is suggested. Pinealectomized animals showed 24-h changes in nucleolar diameters, but these changes differed from those of nonoperated controls in their temporal and phase relations. For example, there was phase advance in A-cells in the juxtacortical area and desynchronization in N-cells. Furthermore, rhythm amplitude was slightly increased in A-cells and slightly decreased in N-cells after pinealectomy. A- and N-cells differed from each other consistently and significantly in the quantitative morphology of their nuleoli and nuclei, and slightly in the patterns of their time-of-day changes in nucleolar size. Nucleolar size usually showed a tendency for a regional gradient, decreasing slightly from periphery toward the center. These observations contribute to our understanding of the chronobiology and functional relations of adrenomedullary chromaffin cells, and of the effects of pinealectomy and sham operations upon this system.

Quantitation of ultrastructural twenty-four-hour changes in pineal nuclear dimensions.

An ultrastructural stereological analysis was made of the nucleus in pinealocytes of 28 male Fischer rats sampled at seven times in a light:dark 12:12 photoperiod cycle. Comparisons of the data from the seven times, in relation to daily means, showed variations in mean nuclear cross-sectional surface area of +/- 16%, and in mean nuclear perimeter of +/- 10%. Peaks in both nuclear dimensions occurred at the middle of the light period and were coincident with the presence of elaborate nuclear configurations and deep indentations. At 2330 and 0430 (1 and 6 h after lights off) nuclear folding was generally less complex. Mitochondria were found in close association with the nuclear folds. Forty percent of nucleoli were marginated at all of the sampling times, except at 1130 (1 h after lights on) and at 2330 (1 h after lights off), when 50% and 35%, respectively, were marginated.

Effects of melatonin and control injections on pineal serotonin and norepinephrine: afternoon injections lower serotonin levels thirty-six percent at light-dark transition.

Pineal weight and serotonin (5-HT) and norepinephrine (NE) contents were studied in male Sprague-Dawley rats that were maintained under controlled light:dark conditions (LD 14:10; lights on 0700-2100) and that received daily subcutaneous injections of either melatonin (20 micrograms in 0.1 ml per animal) or the same volume (0.1 ml) of vehicle alone, at one of two times (0800-0900 or 1800-1900). Animals were sacrificied at four times (1000, 1400, 2000, or 2300) on the day after the last of the 7 consecutive d of injection. Pineal glands were quickly weighed and then frozen for 5-HT and NE assay by the Maickel and Miller extraction and fluorescence methods. Pineal NE content showed differences related to time of day, in confirmation of early work. But no effects attributable specifically to melatonin were found. Melatonin also failed to affect pineal 5-HT content significantly. But injection of either melatonin or vehicle at 1800-1900 led to a reduction in 5-HT content averaging 36% when sampled at either 2000 or 2300, and in comparison with animals injected at 0800-0900. It is suggested that a stresslike or zeitgeberlike effect of injections within a critical period at the end of the daily light phase can cause an earlier-than-normal daily fall in pineal 5-HT content.

Seasonal changes in glycogen level and size of pinealocytes of the white-footed mouse, Peromyscus leucopus: a semiquantitative histochemical study.

Glycogen level in and size of pinealocytes of the feral, white-footed mouse Peromyscus leucopus, were studied by a semiquantitative histochemical method to determine whether seasonal changes exist in them under natural conditions, what temporal pattern they exhibit, and whether 24-hour changes in these parameters exist in different seasons, as shown in the laboratory dd-mice. Marked seasonal changes were seen in both glycogen levels and nuclear densities (ANOVA p less than 0.005). The size of pinealocytes at 09:00 to 10:00 showed one peak (and nadir) seasonal change, with the smallest size in winter (December and February) and a larger size in warmer seasons, with the maximum value in July. Glycogen level in pinealocytes at 09:00 to 10:00 showed bimodal seasonal changes, with lower levels in fall and spring and higher levels in winter and summer. In fall, a circadian trend in glycogen level in pinealocytes was seen, with a higher level at the end of the light period. In winter, the glycogen levels were very high at 09:00, 13:00, 17:00, and 21:00 examined and showed dampening of time-of-day differences. On the other hand, the size of pinealocytes followed a time-of-day change (P less than 0.005), being largest at 13:00 and smallest at 21:00. Thus, marked changes in quantitative structure and chemical activities, suggesting changes in functional activity, in pinealocytes were noted especially in severe, cold winter.

Low molecular weight pineal and cerebral fractions affecting insulin secretion in vitro.

Studies were performed to examine the effects of various fractions of pineal gland origin upon rates of immunoreactive insulin release during short-term incubations of pancreatic islets from pinealectomized rats. Fractions of cerebral cortex were employed in control incubations. An ultrafiltrate of pineal extracts containing materials of molecular weight less than or equal to 1000 daltons stimulated insulin release while fractions of greater molecular weight were without effect. The stimulation of insulin release observed with the lower molecular weight pineal fraction was seen with both nonstimulatory (2 mM) and stimulatory (10 and 30 mM) medium glucose concentrations, but was abolished in the presence of 2,4-dinitrophenol (200 microM). Upon further fractionation of the low molecular weight pineal extract, fractions I (estimated molecular weight range 700-1000 daltons) and 11 (estimated molecular weight range 180-700 daltons) exhibited comparable stimulatory effects upon islet insulin release: similar effects were observed with cerebral cortical fractions in these molecular weight ranges. However, pineal fraction III (estimated molecular weight range less than 180 daltons) exhibited a striking inhibitory effect upon rates of insulin release compared to cerebral cortical fraction III. Potassium, dopamine, norepinephrine and epinephrine concentrations in both pineal and cortical tissue fractions were similarly low. We conclude that insulinotropic constituent(s) of similar molecular weight occur in pineal gland and cerebral cortex, and that the pineal insulinotropic activity stimulates active insulin secretion by the islets independently of concomitant stimulation by glucose; however, lower molecular weight insulinostatic constituent(s) are notable only in the pineal. It is suggested that mild hyperinsulinemia seen in pinealectomized rats under some circumstances may occur as a result of loss of the pineal insulinostatic factor(s).