Nuclear localization of catechol-O-methyltransferase in neoplastic and nonneoplastic mammary epithelial cells.

Catechol-O-methyltransferase (COMT) plays both a regulatory and protective role in catechol homeostasis. It contributes to the regulation of tissue levels of catecholamines and catecholestrogens (CEs) and, by blocking oxidative metabolism of catechols, prevents endogenous and exogenous catechols from becoming a source of potentially mutagenic electrophiles. Evidence implicating CEs in carcinogenesis, in particular in the hamster kidney model of estrogen-induced cancer, has focused attention on the protective role of COMT in estrogen target tissues. We have previously reported that treating hamsters with estrogens causes translocation of COMT to nuclei of epithelial cells in the renal cortex, the site of CE biosynthesis and where the cancers arise. This finding suggested that nuclear COMT may be a marker of a threat to the genome by catechols, including CEs. It is postulated that CEs play a role in the genesis of breast cancer by contributing to a state of chronic oxidative stress that is presumed to underlie the high incidence of this disease in the United States. Therefore, here we used immunocytochemistry to re-examine human breast parenchyma for nuclear COMT. In addition to confirming previous reports of cytoplasmic COMT in mammary epithelial cells, we identified nuclear COMT in foci of mammary epithelial cells in histologically normal breast tissue of virtually all control (macromastia) and cancer patients and in breast cancer cells. There was no correlation between tissue histology and the numbers of cells with nuclear COMT, the size of foci containing such cells, or intensity of nuclear COMT immunostaining. The focal nature of the phenomenon suggests that nuclear COMT does not serve a housekeeping function but that it reflects a protective response to an increased local catechol load, presumably of CEs and, as such, that it may be a characteristic of the population of women studied who share the same major risk factor for developing breast cancer, that of living in the industrialized West.

Effects of L-NAME on cerebral metabolic, vasopressin, oxytocin, and blood pressure responses in hemorrhaged rats.

NG-nitro-L-arginine methyl ester (L-NAME; 250 micrograms/5 microliters), an inhibitor of NO synthase, or the vehicle artificial cerebrospinal fluid (aCSF; 5 microliters) was administered intracerebroventricularly to conscious rats hemorrhaged (0.7 ml/min) to a 20% volume depletion. Hypotension was maximal 5 min after hemorrhage ended, with compensatory recovery to basal levels 20 min later, regardless of drug treatment. L-NAME, however, elevated (P < 0.05) blood pressure (vs. aCSF controls) 40-45 min after intracerebroventricular administration. In normovolemic rats, L-NAME produced a significant pressor response and increased plasma levels of vasopressin (VP) and oxytocin (OT). After hemorrhage, both hormone levels increased, but only OT was further enhanced by L-NAME. Thus centrally produced NO tonically inhibits OT and VP secretion under basal normovolemic conditions and selectively inhibits OT release during hypovolemia. Hemorrhage increased the rates of glucose utilization in the neural lobe, indicative of enhanced efferent neural functional activity. L-NAME further enhanced the metabolic activity in the entire hypothalamoneurohypophysial system of hemorrhaged animals. Several other brain structures involved in the regulation of blood pressure and the stress response were also metabolically affected by the hemorrhage and L-NAME.

Effects of central injection of kyotorphin and L-arginine on oxytocin and vasopressin release and blood pressure in conscious rats.

Intracerebroventricular (I.C.V.) administration of an inhibitor of nitric oxide synthase (NOS) increases oxytocin but not vasopressin secretion, in dehydrated rats [38]. Surprisingly, central injection of L-arginine, the substrate for NOS, caused a similar effect. Kyotorphin (L-tyrosyl-L-arginine), a dipeptide formed from L-arginine by kyotorphin synthetase in the brain may mediate this magnocellular response. Therefore, the dose and time responses of hormone release were compared following I.C.V. injection of kyotorphin and L-arginine to conscious rats that were normally hydrated or deprived of water for 24 h. In water-sated rats, both L-arginine and kyotorphin increased blood pressure and plasma glucose levels coincident with elevating circulating levels of oxytocin, but not vasopressin. In dehydrated animals, both L-arginine and kyotorphin increased plasma oxytocin levels with a similar time course but only kyotorphin decreased vasopressin release. D-arginine, like L-arginine, stimulated secretion of oxytocin, indicating a nonstereospecific effect. A kyotorphin receptor antagonist (L-leucyl-L-arginine) given I.C.V. to dehydrated animals elevated plasma oxytocin and prevented the decrease in vasopressin levels after kyotorphin. Thus, kyotorphin, but not L-arginine, appears to attenuate release of vasopressin either directly from magnocellular neurons or indirectly via modulating compensatory reflexes activated by the pressor response. On the other hand, an excess of L-arginine and kyotorphin within the CNS may mimic the stress response by augmenting release of oxytocin and activating the sympathetic nervous system to increase blood pressure and plasma glucose levels.

Role of NO on vasopressin and oxytocin release and blood pressure responses during osmotic stimulation in rats.

NG-nitro-L-arginine methyl ester (L-NAME, 250 micrograms/5 microliters), an inhibitor of nitric oxide (NO) synthase, or artificial cerebrospinal fluid (5 microliters) was administered intracerebroventricularly to conscious naive rats or to rats treated subcutaneously (15 microliters/kg) with NaCl (0.15, 0.45, or 1.0 M) or given a needle prick only. Intracerebroventricular injection of L-NAME increased plasma concentration of vasopressin (VP) and oxytocin (OT) in control naive rats, indicating that NO tonically inhibits basal secretion of both hormones during isosmotic isovolemic conditions. Osmotic stimulation with hypertonic saline (0.45 and 1.0 M NaCl) elevated plasma levels of both hormones as expected. Central blockade of NO synthase further enhanced secretion of OT during mild, but not strong, osmotic stimulation, whereas the high levels of VP remained unaffected by L-NAME. In animals treated with the needle prick or 0.15 M NaCl, only OT levels were increased after L-NAME. Therefore, NO selectively inhibits OT release in response to a painful stimulus (needle prick) and moderate osmotic stimulation to promote a preferential release of VP. A transient pressor response was observed after subcutaneous injection of 0.15 and 0.45 M NaCl, but a sustained response was obtained after 1.0 M NaCl. Regardless of whether the animals received NaCl solutions, however, treatment with L-NAME elevated blood pressure in all animals. Thus NO-induced vasodilation maintains basal arterial blood pressure and limits the pressor response to osmotic stimulation.

Osmoregulation of the magnocellular neuroendocrine system during lactation.

Glucose utilization and Fos expression were used to compare responses of cerebral structures involved in osmoregulation in virgin and lactating rats given 0.15, 0.85, or 1.5 M NaCl subcutaneously. In virgin animals, glucose utilization increased (P < 0.05) in the supraoptic nuclei (SON), paraventricular nuclei (PVN), and neural lobe (NL) proportionally to the osmotic stimulus (0.15 M NaCl < 0.85 M NaCl < 1.5 M NaCl), whereas metabolism in the median preoptic nucleus (MPO) and median eminence (ME) increased only after 1.5 M NaCl. In lactating rats, enhanced utilization of glucose in response to osmotic stimulation was absent in the PVN (0.85 M NaCl), MPO, and ME or significantly (P < 0.01) reduced (SON, PVN, NL) compared with virgin animals. Glucose utilization in each structure correlated linearly with plasma osmolality but with a lower slope (P < 0.05) in lactating animals. Magnocellular neurons expressing Fos in the SON increased linearly with plasma osmolality and were more numerous (P < 0.05) in control lactating animals but increased less (P < 0.05) than in virgin rats after 0.85 M NaCl. The attenuated magnocellular response during lactation results from reduced afferent activation from osmosensitive forebrain sites.

Crosstalk in the magnocellular system during osmotic stimulation of one supraoptic nucleus.

Neural connections linking the four magnocellular nuclei, i.e., the paired supraoptic (SON) and paraventricular (PVN) nuclei, may contribute to the simultaneous and parallel changes in firing patterns of oxytocinergic neurons during reflex milk ejection. To investigate these neural connections in the absence of suckling, intranuclear release of oxytocin (OT) was stimulated by microdialysis of hypertonic CSF containing 1 M NaCl (HS-CSF) into the right SON area and glucose metabolism of both SONs and PVNs and the neural lobe of virgin and lactating (10-12 day) rats was mapped by the autoradiographic [14C]deoxyglucose (DG) method. OT in the microdialysates and in plasma, obtained before and after 80-90 min of dialysis with CSF or HS-CSF, was quantified by RIA. In both virgin and lactating rats, microdialysis of HS-CSF unilaterally into the SON area significantly (p < 0.05) increased release of OT in the nucleus and into plasma, which was associated with enhanced (p < 0.05) metabolic activity in the ipsilateral and contralateral SON and the neural lobe but not in either PVN. Compared with virgins, lactating rats were less active, had lower (p < 0.05) glucose utilization in the hypothalamo-neurohypophysial system, and less (p < 0.05) OT in plasma during microdialysis of HS-CSF into the SON area. The osmotic stimulus did not activate neural structures (suprachiasmatic and medial amygdaloid nuclei) near the SON in either hemisphere. Thus, neural connections or, less likely, transport of OT via the subarachnoid space, may function to recruit activation of cells in the contralateral SON following hypertonic stimulation of cells in the other SON.