Dennis D. Rasmussen

GnRH Release from the Mediobasal Hypothalamus: in vitro Inhibition by Corticotropin-Releasing Factor

The effects of corticotropin-releasing factor (CRF) on GnRH release from the adult male rat mediobasal hypothalamus (MBH) and median eminence (ME) were studied in an in vitro incubation system. CRF induced a dose-related inhibition of GnRH release from both the MBH and the isolated ME, and this inhibition was blocked by treatment with CRF receptor antagonist. Moreover, GHRF, a hypothalamic peptide similar in size to CRF, did not alter the ME release of GnRH. These results demonstrate that CRF can inhibit in vitro release of GnRH by a CRF receptor-mediated mechanism at the level of the neurosecretory terminals in the ME. Thus, increased hypothalamic CRF secretion associated with stress and adrenalectomy may inhibit hypothalamic GnRH release and produce the suppression of pituitary luteinizing hormone secretion which occurs under these conditions.

Pulsatile Gonadotropin-Releasing Hormone Release from the Human Mediobasal Hypothalamus in vitro: Opiate Receptor-Mediated Suppression

An in vitro perifusion system was used to investigate pulsatile gonadotropin-releasing hormone (GnRH) release from the fetal (20-23 weeks of gestation) and adult human mediobasal hypothalamus (MBH). Fetal human MBHs released GnRH in discrete pulses, with a periodicity of approximately 1 h. Adult human MBHs also released GnRH in a pulsatile manner, with a periodicity of 60-100 min. The calcium-dependent pulsatile GnRH release from fetal human MBHs was suppressed by addition of morphine (10 microM) to the perifusion medium, and this suppression was reversed by addition of the opiate receptor antagonist naloxone (10 microM). These results indicate that the human hypothalamic GnRH pulse-generating mechanism is located entirely within the MBH, and that this pulse generator can maintain intrinsically pulsatile GnRH release independent of all innervation from outside this site. Our data also demonstrate that human hypothalamic pulsatile GnRH release can be suppressed by an opiate receptor-mediated mechanism located within the MBH.

INTRINSIC PULSATILITY OF ACTH RELEASE FROM THE HUMAN PITUITARY IN VITRO

An in‐vitro perifusion system was used to investigate spontaneous ACTH release from human fetal (21–23 weeks gestation) and adult pituitaries. The pattern of ACTH release from fetal pituitaries (n= 7) exhibited a remarkable pulsatile character with a mean (± SEM) pulse interval of 11.3 ± 0.8 min. The mean pulse amplitude was 49.7 ± 6.3 pg, with a nadir to peak increment of 90.7 ± 10.4%. The mean ACTH release rate was 87.2±13.3 pg/2 min. Addition of the calcium chelator EGTA (4 nM) to the perifusion medium induced a significant (p±0.01) decrease in both ACTH release rate (from 102.0 ± 8.5 to 52.0 ± 9.9 pg/2 min) and ACTH pulse amplitude (from 57.7 ± 2.8 to 31.3 ± 4.6 pg) (n= 3). Administration of either 2 nM corticotrophin releasing factor (CRF) or 56 mM KCl induced 10‐ and 2‐fold increases in ACTH secretion, respectively (n= 2). Quarters of adult human pituitaries (n– 6) also secreted ACTH in a pulsatile fashion, with a pulse interval of 14.8±1.7 min, pulse amplitude of 86.7± 10.0 pg, nadir to peak increment of 84.5 ± 9.8%, and overall release rate of 167.2 ± 8.8 pg/2 min. These studies demonstrate that ACTH release from the isolated human pituitary in vitro is characterized by high frequency/low amplitude pulses, independent of hypothalamic stimulation. Accordingly, this spontaneous calcium‐dependent pulsatile ACTH release apparently reflects the activity of an intrinsic intrapituitary pulse‐generating mechanism.

GnRH Release from the Mediobasal Hypothalamus

The effects of oxytocin (OT) on gonadotropin-releasing hormone (GnRH) release from the adult male rat mediobasal hypothalamus and median eminence were studied in an in vitro incubation system. OT indu

Intrinsic pulsatility of luteinizing hormone release from the human pituitary in vitro

An in vitro perifusion system was used to investigate the spontaneous luteinizing hormone (LH) release from 10 human fetal (21–23 weeks of gestation) and 1 adult female pituitaries. The pattern of LH

Episodic gonadotropin-releasing hormone release from the rat isolated median eminence in vitro

Several studies have demonstrated that the rat isolated mediobasohypothalamus can release gonadotropin-releasing hormone (GnRH) in a pulsatile manner in vitro. We have now used an in vitro perfusion system to characterize GnRH release from the adult male Sprague-Dawley rat isolated median eminence (ME) alone. After 1 h stabilization periods, eight MEs released GnRH in an episodic pattern during 4-hour experiments (samples collected every 4 min, RIA in triplicate) with mean (+/- SE) release rates of 2.2 +/- 0.2 pg GnRH/4 min and peak amplitudes of 1.3 +/- 0.2 pg/4 min. The intervals between significant peaks were variable, although 70% were in the range of 12-24 min, i.e. similar to the non-gonadal steroid-modulated frequency of pulsatile luteinizing hormone secretion in gonadectomized rats in vivo. During nine additional experiments, MEs were perifused with control medium (containing 2.5 mM calcium) through the first hour of sample collection, then either calcium-free medium containing 0.5 mM of the calcium chelator EGTA and 100 microM of the intracellular calcium antagonist TMB-8 or calcium-free medium containing just 100 microM TMB-8 for 2 h, followed by a final hour with control medium. Removal of calcium activity decreased the frequency of GnRH peaks from 3.2 +/- 0.3 to 1.3 +/- 0.2/h, which then increased to 2.3 +/- 0.3/h with subsequent replacement of control medium. Removal of available calcium also decreased mean GnRH release by 51 +/- 11%, which returned to basal levels with subsequent calcium replacement.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurosecretion of Human Hypothalamic Immunoreactive β-Endorphin: In vitro Regulation by Dopamine

An in vitro perifusion system was used to investigate immunoreactive beta-endorphin (beta-END-I) release from adult human hypothalami in response to dopamine (DA) and the DA receptor antagonist haloperidol (HAL). Administration of a 1 microM pulse of DA consistently elicited a mean (+/- SE) 88 +/- 9% increase (p less than 0.05, n = 5) in beta-END-I release, whereas 1 microM HAL had no effect. Administration of 1 microM DA during three perifusions in which 1 microM HAL was added to the medium failed to alter basal beta-END-I release. In contrast, DA did evoke an acute 230 +/- 31% increase (p less than 0.05) in beta-END-I release during three matching perifusions with medium containing the alpha-adrenergic antagonist phentolamine. These studies demonstrate that DA can stimulate in vitro release of beta-END-I from the adult human hypothalamus by a DA receptor mediated mechanism.

Role of oxytocin in the modulation of ACTH release in women.

To determine if oxytocin (OT) may have a modulatory role on corticotropin-releasing factor (CRF) and vasopressin (AVP) mediated ACTH-cortisol release in women, serial experiments were performed in which saline, OT, AVP and CRF were administered singly or in combinations. OT administration (2 IU intravenous bolus followed by 111 mIU/min infusion for 3 h) maintained a circulating concentration of 7.7 X 10(-8) M and did not significantly influence basal, AVP or CRF-induced ACTH-cortisol release. In contrast, OT inhibited significantly the potentiating effect of AVP on CRF-stimulated ACTH-cortisol release. These findings suggest that OT and AVP may modulate, in a reciprocal fashion, the CRF-mediated ACTH release and support the contention that OT may be involved in the neuroendocrine response to stress in women.

Pituitary responses to synthetic corticotropin-releasing hormone: Absence of modulato effects by estrogen and progestin

To determine if the activity of the adrenocorticotropic hormonal-adrenal axis is modulated in part by estrogens and progestins, we have compared pituitary and adrenal responses to corticotrophin-releasing hormone in normal women during the early follicular, late follicular, and midluteal phases of the menstrual cycle and in four women undergoing ovariectomy who received estradiol (E2) implants alone or in combination with oral medroxyprogesterone acetate administration. Basal adrenocorticotropic hormone and cortisol levels (9:00 AM) and responses to ovine CRH were unaffected by variations in E2 and progesterone during each phase of the menstrual cycle. In women undergoing ovariectomy who received E2 replacement therapy, basal concentrations of adrenocorticotropic hormone and cortisol (9:00 AM) and the responses to human corticotrophin-releasing hormone were not altered by administration of E2 alone or E2 with medroxyprogesterone acetate. Under these experimental conditions, our findings suggest that physiologic changes in E2 and progesterone levels during the menstrual cycle and replacement doses of estrogen and progestin commonly used clinically do not significantly influence basal adrenocorticotropic hormone and cortisol levels or responsiveness to corticotrophin-releasing hormone.

Positive Correlation between Proopiomelanocortin and Tyrosine Hydroxylase mRNA Levels in the Mediobasohypothalamus of Ovariectomized Rats: Response to Estradiol Replacement and Withdrawal

We have investigated putative dopaminergic regulation of opiomelanotropinergic activity in the arcuate/periarcuate mediobasohypothalamus (MBH) by assessing the changes in MBH tyrosine hydroxylase (TH; rate-limiting enzyme in catecholamine synthesis) and proopiomelanocortin (POMC; opiomelanotropin precursor) mRNA levels under conditions in which endogenous tuberinfundibular dopaminergic activity exhibits marked changes. Adult Sprague-Dawley rats were sacrificed at 09.00 and 15.00 h, and individual MBH POMC and TH cytoplasmic mRNA levels were simultaneously quantified by multiplex solution hybridization-RNase protection assay with protected fragments separated by polyacrylamide gel electrophoresis. In ovariectomized (OVX) rats treated for 3 days with low-dose estradiol (E2) implants (resulting in 18 +/- 4 pg E2/ml serum), the MBH levels of POMC and TH mRNAs were approximately 17 and 31% lower than those measured in OVX controls, respectively. In OVX rats implanted for 20 days with larger E2 implants (99 +/- 9 pg E2/ml serum), POMC and TH mRNA levels were approximately 29 and 41% lower than in OVX controls, respectively. Additional groups were exposed to the higher E2 dose for 20 days and then killed 10 or 20 days after removal of the E2 implant. In these rats, POMC mRNA levels rebounded to the same level seen in OVX controls, while TH mRNA levels even exceeded control values by 22-27%. TH and POMC mRNA levels did not change significantly between 09.00 and 15.00 h, except 10 days after removal of the E2 implants, when 09.00 h POMC mRNA levels were higher than the 15.00 h levels. MBH POMC and TH mRNA levels were positively correlated with each other within individual animals. This correlation is maintained when both POMC and TH mRNA levels are suppressed in response to both 3-day low-dose and 20-day high-dose E2 treatment. However, although rat MBH opiomelanotropinergic and tuberoinfundibular dopaminergic mRNA biosynthesis thus appear to be positively correlated, the coregulation or functional interactions of these two neuronal systems remain to be determined.