Microdialysis without acetylcholinesterase inhibition reveals an age-related attenuation in stimulated cortical acetylcholine release

Aging-related differences in the ability of cortical cholinergic inputs to respond to local depolarization was assessed in young (3-6 months) and old (26-33 months) awake rats using in vivo microdialysis in the absence of an inhibitor of acetylcholinesterase. Rats were perfused, using a within-subjects, repeated session design, with vehicle (aCSF) or K(+) (25, 50, 100 mM). Perfusion of K(+) resulted in a dose-dependent increase in cortical ACh efflux with comparable efflux seen between the two ages following 25 mM (50%) and 50 mM (100%) K(+). In contrast, aged rats exhibited a marked attenuation (330%) in ACh efflux relative to young adult rats (650%). These data reveal aging-related decreases in the responsiveness of cortical cholinergic afferents, tested under physiologically relevant conditions, to local depolarization and may provide a neuronal mechanism contributing to the cognitive deficits reported in normal aging- and age-related pathological conditions.     

Motherhood alters the cellular response to estrogens in the hippocampus later in life

Although controversial, estrogen replacement therapy has been implicated as a possible therapeutic agent for ameliorating age-related cognitive decline in postmenopausal women. We have shown previously that different types of estrogen promote hippocampal neurogenesis in a dose-dependent manner in young adult female rats. However, previous studies have not found a beneficial effect of 17β-estradiol in middle-aged female rats. The aim of the present study was to determine the acute effects of 17β-estradiol, 17α-estradiol, and estrone on hippocampal cell proliferation in middle-aged ovariectomized female rats and to determine whether effects are dependent on previous reproductive experience. Middle-aged multiparous female rats or age-matched virgin female rats were injected subcutaneously with vehicle or 10 μg dose of 17β-estradiol, 17α-estradiol, or estrone, and then given BrdU 30 min later and perfused 24 h later to assess cell proliferation. All estrogens significantly upregulated cell proliferation in the hippocampus in middle-aged multiparous females but none of the estrogens upregulated cell proliferation in the middle-aged virgins. Therefore, previous reproductive experience may make the older brain more responsive to estrogens later in life. We also found that 17α-estradiol upregulated cell proliferation to a greater degree than the other estrogens in the multiparous females. Together these findings may lead to the development of new therapeutic advances in the treatment of symptoms associated with menopause in women.     

Conformational changes in muscarinic receptors may produce diminished cholinergic neurotransmission and memory deficits in aged rats

Both clinical and laboratory studies suggest that age-related memory deficits may be due, at least in part, to disturbances in muscarinic acetylcholine (mAChR) receptors. In order to further evaluate this premise, the present studies examined the electrophysiological responses rates of hippocampal pyramidal cells to iontophoretically applied ACh in young, middle-age and aged animals. The relationship between age and muscarinic agonist and antagonist binding in the hippocampus was also examined. In addition, possible age-related changes in receptor-effector coupling were assessed by determining calmodulin levels and the activities of phospholipid methyltransferase I and II. Analysis of electrophysiological data showed selective age-related decrements in the ability of ACh to alter burst rate but not simple spike rate. These age-related decreases in the efficacy of ACh to increase burst rate were not paralleled by decreases in mAChR density as assessed by ³H-QNB binding, but they were temporally paralleled by age-related changes in the ability of oxotremorine to inhibit ³H-QNB binding. In the young animals, the resultant Hill coefficients derived from these analyses approached 1, while in the middle and old aged animals, the Hill coefficients deviated significantly from 1, indicating the possible existence of 2 or more receptor states with differential affinity for oxotremorine in the 2 older age groups. When carbamylcholine was used to inhibit ³H-QNB, these complex binding patterns were seen even in the young, since carbamylcholine induces conformational/orientational changes in the mAChR while oxotremorine does not. It is suggested that declines in mnemonic ability that have been reported previously, parallel the age-related conformation/orientational changes observed in the mAChR since these changes result in a receptor that is “neurophysiologically desensitized.”     

Conformational changes in muscarinic receptors may produce diminished cholinergic neurotransmission and memory deficits in aged rats

Both clinical and laboratory studies suggest that age-related memory deficits may be due, at least in part, to disturbances in muscarinic acetylcholine (mAChR) receptors. In order to further evaluate this premise, the present studies examined the electrophysiological responses rates of hippocampal pyramidal cells to iontophoretically applied ACh in young, middle-age and aged animals. The relationship between age and muscarinic agonist and antagonist binding in the hippocampus was also examined. In addition, possible age-related changes in receptor-effector coupling were assessed by determining calmodulin levels and the activities of phospholipid methyltransferase I and II. Analysis of electrophysiological data showed selective age-related decrements in the ability of ACh to alter burst rate but not simple spike rate. These age-related decreases in the efficacy of ACh to increase burst rate were not paralleled by decreases in mAChR density as assessed by ³H-QNB binding, but they were temporally paralleled by age-related changes in the ability of oxotremorine to inhibit ³H-QNB binding. In the young animals, the resultant Hill coefficients derived from these analyses approached 1, while in the middle and old aged animals, the Hill coefficients deviated significantly from 1, indicating the possible existence of 2 or more receptor states with differential affinity for oxotremorine in the 2 older age groups. When carbamylcholine was used to inhibit ³H-QNB, these complex binding patterns were seen even in the young, since carbamylcholine induces conformational/orientational changes in the mAChR while oxotremorine does not. It is suggested that declines in mnemonic ability that have been reported previously, parallel the age-related conformation/orientational changes observed in the mAChR since these changes result in a receptor that is “neurophysiologically desensitized.”     

Age-related increases in LPS-stimulated nitric oxide production from cultured rat bone marrow cells

Objective: To understand bone metabolism during senescence, we examined age-related change in nitric oxide (NO) production from bone marrow cells stimulated by lipopolysaccharide (LPS). Methods: We evaluated the age-related change in the NO production and expression of iNOS protein and mRNA of LPS-stimulated bone marrow cells collected from the tibiae of young and retired female and young and retired male rats. In addition, we used flow cytometry to assess changes in the distribution of CD14, a cell surface receptor of LPS. Results: The results revealed that NO production from bone marrow cells stimulated with LPS changed with aging. The NO levels in old rats were significantly higher (P<0.05) than those in young rats. Polymerase chain reaction (PCR) analysis indicated that the LPS-induced expression of iNOS mRNA was augmented in retired rats. Although the distribution pattern of the bone marrow cells was similar between young and retired rats, the percentage of CD14-positive cells in specific populations differed between the age groups. Specifically, in the granule-containing bone marrow cells, the percentage of CD14-positive cells was increased in retired rats. Conclusion: Our results indicate that LPS-stimulated NO production from rat bone marrow cells increased with age and that the difference in responsiveness might be due to changes in the percentage of CD14-positive cells in the bone marrow.     

Effects of old age on human skeletal muscle energetics during fatiguing contractions with and without blood flow

We recently reported lower glycolytic flux (ATP_GLY) and increased reliance on oxidative ATP synthesis (ATP_OX) in contracting muscle of older compared to young humans. To further investigate this age-related difference in the pathways of ATP synthesis, we used magnetic resonance spectroscopy to determine the rates of ATP_OX, ATP_GLY and net phosphocreatine hydrolysis in vivo during maximal muscle contractions under free-flow (FF) and ischaemic (ISC) conditions in the ankle dorsiflexors of 20 young (27 ± 3 years; 10 male, 10 female) and 18 older (70 ± 5 years; 10 male, 8 female) adults. We hypothesized that ATP_GLY would be higher in young compared to old during FF contractions, but that old would be unable to increase ATP_GLY during ISC to match that of the young, which would suggest impaired glycolytic ATP synthesis with old age. Peak glycolytic flux during FF was lower in older (0.8 ± 0.1 m m ATP s⁻¹) compared to young (1.4 ± 0.1 m m ATP s⁻¹, P < 0.001) subjects. During ISC, peak ATP_GLY increased in old to a level similar to that of young (1.4 ± 0.2 m m ATP s⁻¹, 1.3 ± 0.2 m m ATP s⁻¹, respectively; P = 0.86), suggesting that glycolytic function remains intact in aged muscle in vivo . Notably, older adults fatigued less than young during both FF and ISC ( P ≤ 0.004). These results provide novel evidence of unimpaired in vivo glycolytic function in the skeletal muscle of older adults during maximal isometric dorsiflexion, and suggest a potential role for differences in metabolic economy and as a result, metabolite accumulation, in the fatigue resistance of the old.     

Diabetes mellitus and responses of the urinary bladder to acetylcholine: an in vitro study

This study was prompted by the inconsistent reports and apparent controversies that exist in the biomedical literature on the responses of diabetic bladder strips to cholinergic nerve stimulation or exogenously-administered muscarinic agonists, especially acetylcholine (ACh). In the present study, acetylcholine-induced contractions of urinary bladders isolated from normoglycaemic (normal) and streptozotocin-treated, diabetic Wistar rats were examined under physiological conditions. Mechanical contractile changes of the isolated urinary bladders of STZ-treated, diabetic rats in response to bath-applied acetylcholine were compared with those obtained from isolated urinary bladders of normal, age-matched, control rats. Results obtained show that urinary bladders from diabetic rats were always more spontaneously active after mounting, than those of the age-matched normal, control rats. ACh (10(-8)-10 (-4) M) provoked concentration-related, atropine-sensitive contractions of the isolated urinary bladders of both diabetic and age-matched normal, control rats. However, acetylcholine always induced more powerful and greater contractions of the diabetic bladders compared with bladders from the age-matched normal, control rats. The magnitude and/or intensity of the diabetic bladder enhanced contractile responses to ACh continued to increase as the diabetic state of the animals progressed.     

Age-dependence of effects of A1 adenosine receptor antagonism in rat hippocampal slices.

1. Population excitatory postsynaptic potentials (EPSPs) and population spikes evoked in area CA1 of hippocampal slices from aged Fischer 344 rats were significantly smaller in amplitude than responses obtained in slices from young Fischer 344 rats. 2. The A1 adenosine receptor antagonist 8-cyclopentyltheophylline (8-CPT) produced a concentration-dependent increase in synaptic potentials in slices from both young and aged rats. Low concentrations (1 nM) of 8-CPT were effective in producing increases in both population spike amplitudes and population EPSP slopes in young and aged rat slices. Response increases were maximized by 100 nM 8-CPT in slices from rats of both age groups. 3. Adenosine antagonism produced greater average increases in synaptic responses in hippocampal slices from aged rats at all concentrations tested (1.0 nM-1.0 microM). A qualitative age-related difference in the response to 8-CPT was also observed; 8-CPT produced a late component, consisting of multiple population spikes, in evoked responses in slices obtained from aged but not young rats. 4. Adenosine antagonism significantly increased the maximum evocable response (both spike amplitude and EPSP slope) in slices from aged rats, relative to increases observed in slices from young rats. This suggested that smaller synaptic potentials seen in slices from aged rats were in part due to greater levels of "tonic" adenosinergic inhibition. 5. Slices from young and aged rats were incubated in the adenosine reuptake inhibitor soluflazine (R64719; 1.0, 10, and 100 microM) and the inhibition of population EPSPs was observed for 60 min. No difference was observed in the rate of inhibition or the maximal level of inhibition produced by soluflazine, in slices from rats of either age group. 6. Application of (+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]cyclo-hepten- 5,10-imine hydrogen maleate (MK-801) and 2-amino-5-phosphonopentanoic acid (2-AP5), antagonists of N-methyl-D-aspartate (NMDA) excitatory amino acid (EAA) receptors, reduced the late multiple population spike component in slices from aged rats incubated in 8-CPT. A smaller direct effect of the NMDA antagonists was observed in slices from aged rats in the absence of 8-CPT treatment at maximal response levels. No effect of NMDA receptor antagonism was observed in slices from young rats under either condition. 7. Hippocampal tissue, from young and old rats utilized in the electrophysiological experiments, was assayed for A1 adenosine binding site density with a saturating concentration of radiolabeled agonist and antagonist. Guanine nucleotide modulation of agonist binding was also measured.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of age and stress on regional noradrenaline metabolism in the rat brain

Levels of noradrenaline (NA) and its major metabolite, 3-methoxy-4-hydroxyphenylethyleneglycol sulfate (MHPG-SO₄), were determined in eight brain regions of non-stressed rats at 2, 10 and 15 months of age, and of rats at 2 and 15 months of age stressed by immobilization for 3 hours. The NA levels in older rats were significantly lower in the hypothalamus, pons+med.obl. and midbrain, and higher in the amygdala, thalamus, hippocampus and cerebral cortex as compared to those of 2 month old rats. The MHPG-SO₄, levels in the older rats were significantly lower in the hypothalamus, amygdala, pons+med.obl. and midbrain, and higher only in the cerebral cortex than those in 2 month old rats. Immobilization stress caused significant increases in NA turnover in all brain regions of both 2 and 15 month old rats. Age-related difference in the degree of stress-induced change in NA metabolism was found only in the hypothalamus; the increase of MHPG-SO₄ by stress was greater in 2 month old rats than in 15 month old rats, although both age groups of rats showed the same degree of NA reduction by stress. These data suggest that brain NA metabolism changes in an age-related fashion, and that apparent regional differences exist in the pattern of these changes. Specifically, it appears that there is an age-related difference in the response of noradrenergic neurons to stress in the hypothalamus.     

Exposure to young induces postpartum-like fighting in virgin female mice

The study was designed to determine whether stimulation derived by adult virgin female mice from 1-day old pups is capable of inducing aggressive behavior similar to that displayed by lactating mice. The first experiment showed that virgins maintained continuously in the presence of pups attacked an adult male mouse intruder. It also was demonstrated that this fighting was not displayed when testing occurred 3 hr following removal of the foster young. Fighting was reinstated when the animals were re-tested 30 min following the reintroduction of the young. A final experiment showed that, in contrast to the virgin females maintained with young that did not fight, virgins that fought were rated as evidencing a significant amount of nipple development. It was concluded that nipple stimulation may be a significant condition for the production of fighting behavior in female mice.     

Phosphatidylserine increases acetylcholine release from cortical slices in aged rats

Acetylcholine release was investigated in cortical slices superfused with choline-enriched Krebs solution containing physostigmine. Slices were prepared from 3 and 24 month old rats treated with either Tris buffer or sonicated suspensions of phosphatidylserine and phosphatidylcholine in Tris buffer. Slices were electrically stimulated at frequencies of 1, 2 and 5 Hz for 5 min periods preceded and followed by rest periods. ACh content of the superfusate was quantified by bioassay. In the 24 month old rats treated with Tris buffer, acetylcholine release, at all frequencies tested, was approximately 50% lower than that in the 3 month old rats. On the contrary, no significant decrease in ACh release was found in the 24 month old rats treated for 30 days with phosphatidylserine (15 mg/kg IP). The same treatment did not increase acetylcholine release in 3 month old rats. Acetylcholine release in 24 month old rats receiving a single administration of phosphatidylserine (15mg/kg IP) or phosphatidylcholine (15 mg/kg IP) for 30 days was as low as in the 24 month old rats receiving the Tris buffer only. It is proposed that the chronic phosphatidylserine treatment may reduce the age-induced decrease in acetylcholine release by acting on the stimulus-secretion coupling mechanism.     

Phosphatidylserine increases acetylcholine release from cortical slices in aged rats

Acetylcholine release was investigated in cortical slices superfused with choline-enriched Krebs solution containing physostigmine. Slices were prepared from 3 and 24 month old rats treated with either Tris buffer or sonicated suspensions of phosphatidylserine and phosphatidylcholine in Tris buffer. Slices were electrically stimulated at frequencies of 1, 2 and 5 Hz for 5 min periods preceded and followed by rest periods. ACh content of the superfusate was quantified by bioassay. In the 24 month old rats treated with Tris buffer, acetylcholine release, at all frequencies tested, was approximately 50% lower than that in the 3 month old rats. On the contrary, no significant decrease in ACh release was found in the 24 month old rats treated for 30 days with phosphatidylserine (15 mg/kg IP). The same treatment did not increase acetylcholine release in 3 month old rats. Acetylcholine release in 24 month old rats receiving a single administration of phosphatidylserine (15mg/kg IP) or phosphatidylcholine (15 mg/kg IP) for 30 days was as low as in the 24 month old rats receiving the Tris buffer only. It is proposed that the chronic phosphatidylserine treatment may reduce the age-induced decrease in acetylcholine release by acting on the stimulus-secretion coupling mechanism.     

Age-related changes in power output during repetitive contractions of rat medial gastrocnemius muscle

Changes in isometric force, power output and relaxation rate have been measured during repetitive tetanic contractions in 2 groups of rats of different ages. During the first 5 contractions there were no differences between a young and mature group. In contrast to isometric force production, which decreased about 3% per contraction, power output initially increased to 108% of the power output in the first contraction. A greater reduction in power output and relaxation rate after the 5th contraction indicated a greater reduction of the cross-bridge cycling rate in the younger rats. ATP, phosphocreatine and lactate concentrations after the last contraction were not different between the age-groups. In contrast IMP production, which has been suggested may play a regulatory role during fatigue was twice as high in the young rats. Judged by isometric force production there is no age-related difference in fatiguability. However, profound differences were observed in power output, which indicates that quantification of fatigue as a loss of isometric force may be seriously misleading when considering the functional status of the muscle for normal dynamic contractions.     

Effects of aging and phenobarbital on the rat liver microsomal drug-metabolizing system

Significant declines in the non-induced activities of liver microsomal drug-metabolizing enzymes and in the amount of cytochrome P-450 occur between maturity (16 months) and senescence (27 months) in male Fischer 344 rats, whereas there are essentially no differences between very young (1 month) and mature animals. Several hepatic responses to chronic phenobarbital administration also demonstrate marked age-dependent changes. The livers of young and mature animals exhibit: (1) greater hepatomegaly; (2) faster rates of induction and post-induction recovery of microsomal mixed function oxidase enzyme activities and hemoprotein concentration; and (3) higher maximally induced levels of these components in comparison to senescent rats. When considered with information from previous studies, the present data suggest that the age-related decline in liver drug metabolism may be due to qualitative and/or quantitative changes in the structural and/or functional components of the hepatic microsomal mixed function oxidase system.     

Developmental Trajectories of Neural Mechanisms Supporting Conflict and Error Processing in Middle Childhood

To study age-related changes in cognitive control, event-related potentials (ERPs) of children aged 6–9 years, 10–12 years, and young adults were recorded during a Flanker task. Younger children were more susceptible to conflict than older ones and adults. The N2 in incongruent trials was smaller in younger than older children, who did not differ from adults. The error-related negativity (ERN) following errors, however, was larger in adults than in children, but did not differ between younger and older children. Delayed development of neural responses to error processing relative to conflict monitoring may indicate dissociable maturational time tables of subdivisions of the anterior cingulate cortex.     

Developmental trajectories of neural mechanisms supporting conflict and error processing in middle childhood

To study age-related changes in cognitive control, event-related potentials (ERPs) of children aged 6-9 years, 10-12 years, and young adults were recorded during a Flanker task. Younger children were more susceptible to conflict than older ones and adults. The N2 in incongruent trials was smaller in younger than older children, who did not differ from adults. The error-related negativity (ERN) following errors, however, was larger in adults than in children, but did not differ between younger and older children. Delayed development of neural responses to error processing relative to conflict monitoring may indicate dissociable maturational time tables of subdivisions of the anterior cingulate cortex.     

Interaction between hypersomatotropism and age in the Wistar-Furth rat

The purpose of this study was to examine body growth parameters and serum somatomedin levels in progressively older rats exposed to high levels of circulating growth hormone (GH). The effects of growth hormone secreting tumors arising from GH3 cell injection were evaluated in 3, 5, 9, 18 and 24 month old female Wistar-Furth rats. In all tumor-bearing rat age groups, body weights were in excess of 70% of age-matched controls. Contributing to these body weight gains were significant increases in heart, liver, kidney and hind limb muscle weights. Fat pads from the tumored groups were similar to controls in the young (3, 5, 9 month) rats but were significantly smaller in the older groups at 18 and 24 months. Elevated GH predictably increased somatomedin levels in young rats while in the 18 and 24 month old groups, smaller increases were noted. These results show that old rats respond to GH in a mechanism involving somatomedin and that significant increases in somatic growth can be obtained, even at advanced age.     

ACh-Evoked complex membrane potential changes in mouse submaxillary gland acini

The responses in membrane potential and resistance of acinar cells to iontophoretically applied acetylcholine (ACh) were investigated using intracellular micro-electrode recording in superfused segments of mouse submaxillary gland. For measurements of membrane resistance and acetylcholine equilibrium potential (E_ACh), two micro-electrodes were inserted into neighbouring communicating cells. Current could be injected through one of the electrodes.The pattern of membrane potential change induced by ACh depended on the resting potential. Simple hyperpolarizations were induced at low resting potentials, while biphasic potential changes (depolarization followed by hyperpolarization) or simple depolarizations were observed at relatively high resting potentials. A similar dependence of the ACh induced potential change on the resting potential was obtained in experiments in which the resting membrane potential was set at different levels by injecting direct current and stimulating the same cell with equal doses of ACh. The ACh equilibrium potential ranged widely between –45 and –75 mV.Under special conditions the conversion in response to ACh from a hyperpolarization to depolarization could be obtained without change in resting potential. Small doses of ACh evoked simple depolarization, while medium doses induced biphasic responses and large doses of ACh caused hyperpolarization. The effect of a low concentration of atropine on the response was an initial block of hyperpolarization followed by a secondary block of depolarization. Intracellular injection of TEA ions converted the ACh induced potential response from hyperpolarization to depolarization. Both the depolarizing and hyperpolarizing ACh responses were accompanied by a marked reduction in membrane resistance. The depolarization was abolished by a severe reduction in external Na concentration, while the hyperpolarization was sensitive to alterations in external K concentration.These results indicate that some of the complex responses in submaxillary gland acinar cells to ACh may be explained by the interaction between two different kinds of potential change (Na dependent depolarization and K dependent hyperpolarization).     

Modulation of reflex responses in activated ankle dorsiflexors differs in healthy young and elderly subjects

During voluntary contractions, motor neurone activity is modulated by descending input and sensory feedback. Impaired excitatory afferent feedback with ageing may, therefore, alter motor control. This study investigated the age-related changes in afferent feedback through the recording of reflex responses during voluntary muscle activation. Short- and long-latency components of the stretch reflex and Hoffmann reflex (H-reflex) were recorded during voluntary contractions (10% of maximal voluntary contraction; MVC) of the ankle dorsiflexor muscles of young and elderly adults (≥70 years). Furthermore, the modulation of spinal reflex excitability was analyzed at different torque levels (10–50% MVC). The short-latency stretch reflex and the H-reflex areas were similar in the two age groups at 10% MVC whereas the area of the long-latency component of the stretch reflex augmented with ageing (P < 0.05). However, the area of the H-reflex increased linearly with the level of contraction up to 50% MVC in young adults, whereas it slightly increased to 30% MVC and plateaued thereafter in elderly adults. The absence of age-related changes in the short-latency stretch reflex and H-reflex areas suggests that the reflex circuitry and the sensitivity of the muscle spindles are not substantially affected by ageing. The modest increase in the H-reflex area with the contraction intensity in elderly adults, however, indicates that the modulation of afferent feedback is reduced with advancing age. This observation, associated with a greater long-latency stretch reflex, suggests that elderly adults rely more on central than peripheral mechanisms to regulate motor output of the dorsiflexor muscles.     

Influence of oestrous cycle and pregnancy on the reactivity of the rat mesenteric vascular bed

In isolated, perfused mesenteric vascular beds from female rats, it was assessed whether the constrictor response to cirazoline, an alpha(1)-adrenergic agonist, or acetylcholine (ACh)-induced relaxation was altered by oestrous cycle or pregnancy and the ability of nitric oxide (NO), prostanoids and endothelium-derived hyperpolarizing factor (EDHF) to modulate these responses. Mesenteries, removed from female rats on each oestrous cycle day and gestation day 16, were perfused with physiological salt solution. Tone was induced with cirazoline (1 micromol/l), and concentration-response curves to ACh generated. Responsiveness to ACh was tested in the presence of N(omega)-nitro-L-arginine (L-NA), ibuprofen (IBU) and tetrabutylammonium (TBA), to inhibit nitric oxide synthase (NOS), cyclo-oxygenase and K(+) channels respectively. Cirazoline-induced tone was smaller in pro-oestrous and pregnant groups, but the increase in tone to L-NA was larger in pregnant compared with oestrous and dioestrous groups. Control responses to ACh were not different, but L-NA attenuated the response in virgin groups only. IBU did not affect the ACh response, but TBA attenuated it in all groups. When TBA was introduced first, ACh-induced dilatation was significantly reduced and not altered by L-NA addition. These results suggest that in the mesenteric vascular bed from cycling and pregnant rats, EDHF is the major mediator of ACh-induced dilatation and NOS may be up-regulated in pregnant and pro-oestrous rats.