Introduction

The adrenal gland is composed of two separate functional regions: The outer cortex is principally responsible for corticosteroid synthesis and secretion. The inner medulla, containing the adrenomedullary chromaffin cells (AMC), synthesizes and secretes catecholamines (i.e., adrenaline, noradrenaline and dopamine), and is perhaps best known for its contribution to the 'fight-or-flight' response. In mature animals, metabolic or physiological stress increases activity in the sympathetic nervous system leading to acetylcholine (ACh) release from splanchnic nerve endings, which innervate AMC. Released ACh activates nicotinic receptors on AMC, resulting in secretion of catecholamines (CA) into the blood. Circulating CA have well-defined roles in the fight-or-flight response of adult animals (Fig. 1), ensuring that adequate blood flow to vital organs (heart and lung) is maintained.

In species that are relatively immature at birth, such as rat and man, sympathetic innervation to several target organs, including the splanchnic projections to the adrenal medulla, are non-functional at birth (25). This suggests that CA secretion from immature adrenal glands is not under nervous regulation. However, despite the lack of neurogenic control of adrenal CA secretion in neonatal animals, several studies have suggested that AMC secrete CA during physiological stressors (e.g., hypoxia), and that the released CA are vital for survival of the neonate during birth and subsequent hypoxic events.

As early as 1961, the oxygen sensitivity of the adrenal medulla was recognized. Comline and Silver (5) demonstrated that asphyxiated fetal sheep had elevated plasma CA and depleted adrenal CA even in the absence of mature sympathetic innervation. In the 1980s, Seidler and Slotkin described a similar non-neurogenic regulation of CA release in the neonatal rat, by demonstrating that prior to maturation of innervation to the adrenal gland, 1 hr of inspired hypoxia (5% 02) depleted adrenal CA. CA release was associated with lung fluid absorption and initiation of surfactant secretion, clamping of the fetal heart rate, and brown fat mobilization (Fig. 1) (25-27). The hypoxia-induced CA release was not dependent upon activation of sympathetic nerves because block of nicotinic receptors with chlorisondamine did not prevent the hypoxia-induced CA surge (25). Seidler and Slotkin (28) also demonstrated that these non-neurogenic responses to hypoxia were absent in adult animals, but that three weeks after splanchnic nerve transection in these animals, the non-neurogenic hypoxia-induced CA secretion returned. Taken together, these studies suggested that AMC may sense hypoxia prior to the maturation of sympathetic innervation and that this 02-sensitivity is lost in mature animals.

Neonatal (Non-innervated)

Immature if: Splanchnic , Neurons

AMC r1"

Catecholamines

Catecholamines

Juvenile (Innervated)

Hypoxia

direct

; Mature lj Splanchnic 'I Neurons

Catecholamines +

Systemic Effects: a-Receptors:

Systemic Effects:

a-Receptors: Cardio-protection (maintained conductance and sinus thythm) P-Receptors: Lung Maturation (fluid absorption and surfactant secretion)

Catecholamines +

Systemic Effects: a-Receptors:

Vasoconstriction (systemic)

Vasodilation (muscular) fi-Receptors: Cardiac output (increased rate)

Figure I. Comparison of the role of hypoxia-induced secretion of catecholamines from adrenomedullary chromaffin cells (AMC) of different ages. Neonatal [postnatal (P) 1 -2 day old] AMC are sensitive to hypoxia and secrete catecholamines in the absence of functional splanchnic innervation. Catecholamines released into the circulation promote neonatal survival during hypoxia via initiation of surfactant secretion in the lung and maintenance of the heart's conduction characteristics. In contrast, juvenile (P13-20) AMC may not respond directly to hypoxia (though some adult cells may), but release catecholamines during splanchnic nerve activation via the central nervous system (CNS). These differences in physiological responses can be considered adaptations of the neonate that contribute to physiological changes associated with the transition to extrauterine life.

2. Catecholamine Release From Neonatal AMC Promotes Survival During Hypoxia

Birth is associated with both fetal hypoxia and hypercapnia due to intermittent occlusions ofthe umbilical cord (15), In mature animals, physiologic adjustments to hypoxia are initiated and maintained by several systems, including ventilatory reflex activation via the carotid body and hypoxic pulmonary vasoconstriction. The transition from fetal to extra-uterine life requires that the lungs be cleared of fluid and that surfactant be secreted to enable proper lung expansion and gas exchange across the alveoli. It is known that CA play a pivotal role in these processes through the activation of p-adrenergic receptors in the lung (34) and a-adrenergic receptors in the heart (27).

Seidler and Slotkin (25) demonstrated that CA derived from the adrenal gland are important for lung maturation after birth, and are vital for neonatal survival during hypoxia. Administration of the P2-blocker ICI-118551 compromised the survival of neonatal rats [postnatal (P) day 1-2] during hypoxia, but did not affect mortality in more mature animals (P14) that had further developed lung function (25). Whereas adrenalectomy dramatically compromised the ability of the neonatal rat to survive hypoxia (5% 02 for 1 hr), interference of CA release from sympathetic nerve endings did not (25). This suggests a vital role for adrenal-derived CA in the hypoxic tolerance of P1 -2 rats. Interestingly, hypoxia in the presence of the cardiac-specific p,-receptor blocker atenolol did not affect neonatal mortality, suggesting that cardiac p-receptors are not involved in tolerance of neonates to hypoxia (27).

The principal cardioprotective effect of adrenal CA is maintenance of heart rate and conduction characteristics (Fig. 1) (27). Administration ofthe a-receptor blocker phenoxybenzamine (PBX) to 1 day-old rat pups during normoxia (21% inspired 02) did not alter cardiac function. However, PBX applied concomitantly with hypoxia caused a marked decline in heart rate, slowing of sinus rhythm, atrioventricular block, and cardiac failure (27, 28). Although mature animals express few cardiac relative to neonatal animals predominantly express the (27). As the neonate matures, cardiac receptors are replaced with the subtype and the cardioprotective effects of adrenal-derived CA disappears (27). The hypoxia-induced secretion of adrenal CA, and the presence ofseveral physiological mechanisms that utilize circulating CA to promote development and survival of neonates can be considered adaptations of the adult stress response that are designed to promote neonatal survival during and after birth.

3. The Oxygen Sensing Mechanism of Chromaffin Cells

The work described above led us to hypothesize that neonatal (P1-2) 'non-

innervated' AMC function as direct sensors of Po2, and that this 02-sensing mechanism is absent in juvenile (P14) cells, which are functionally innervated. To test this hypothesis, we isolated AMC from both age groups, maintained them in short-term cell culture (1-4 days) and determined whether or not they were hypoxia-sensitive using patch clamp recording of whole-cell currents and membrane potential. CA secretion was also measured by HPLC with electrochemical detection.

Figure 2. Comparison of the direct 02-sensitivity of cultured neonatal (panels A, C, and E) and juvenile (panels B, D, and F) adrenal chromaffin cells (AMC). A: Current-voltage plot illustrating the reversible hypoxic suppression of outward currents recorded from neonatal AMC. Hypoxia (H) inhibits currents from the normoxic control (C) level, and the effects are reversible upon washout (W) of the hypoxic solution. B: Hypoxia fails to suppress outward currents in cells from juvenile AMC. The insets in A and B are current traces at a potential of +30 mV from the holding potential of-60 mV. C and D: Hypoxia induces a receptor potential of ~ 15 mV in singly isolated neonatal but not juvenile AMC. E and F: Hypoxia induces catecholamine secretion in cultures of neonatal but not juvenile AMC and secretion is inhibited by the L-type Ca2+ channel blocker nifedipine (Nif). "NE", norepinephrine; "E", epinephrine; "DA", dopamine; "HK", high K+. Panels C-F are reproduced with permission from Ref. 33.

Figure 2. Comparison of the direct 02-sensitivity of cultured neonatal (panels A, C, and E) and juvenile (panels B, D, and F) adrenal chromaffin cells (AMC). A: Current-voltage plot illustrating the reversible hypoxic suppression of outward currents recorded from neonatal AMC. Hypoxia (H) inhibits currents from the normoxic control (C) level, and the effects are reversible upon washout (W) of the hypoxic solution. B: Hypoxia fails to suppress outward currents in cells from juvenile AMC. The insets in A and B are current traces at a potential of +30 mV from the holding potential of-60 mV. C and D: Hypoxia induces a receptor potential of ~ 15 mV in singly isolated neonatal but not juvenile AMC. E and F: Hypoxia induces catecholamine secretion in cultures of neonatal but not juvenile AMC and secretion is inhibited by the L-type Ca2+ channel blocker nifedipine (Nif). "NE", norepinephrine; "E", epinephrine; "DA", dopamine; "HK", high K+. Panels C-F are reproduced with permission from Ref. 33.

3.1. Development of 02 Sensing in AMC

Similar to the mechanism of 02-sensing by the glomus cells of the carotid body (18) and pulmonary arterial myocytes (2), exposure of neonatal AMC to hypoxia (Po2 ~ 5 mmHg) reversibly inhibited outward currents and induced a membrane depolarization (receptor potential) of ~ 15 mV (Fig. 2A, C, and E) (31,

33). Additionally, 1 hr of hypoxia stimulated CA secretion in cultures of neonatal AMC by ~6 x basal, and secretion of all three CA (dopamine, norepinephrine and epinephrine) was enhanced. Hypoxia-induced CA secretion was dependent on extracellular Ca2+ and attenuated by the L-type Ca2+ channel blocker nifedipine (10 jiM) (33). In aparallel study, Mojet et al. (21) demonstrated that the electron transport chain (ETC) inhibitor cyanide (CN; 2.5 mM) mimicked the effects of hypoxia on CA secretion, as detected by carbon fiber electrodes placed adjacent to isolated neonatal AMC. Interestingly, in both studies responses to hypoxia were absent in juvenile AMC (Fig. 2B, D, F).

In contrast to the experiments summarized above, several reports have suggested that adult AMC may express 02-sensing mechanisms. In these studies AMC were maintained in either long-term (7 days) (20) or short-term (1-2 days) (16) culture, or studied in an adrenal slice preparation (30). Hypoxia (-5% 02) suppressed K+ currents, induced membrane depolarization, increased intracellular Ca2+, and enhanced CA secretion in -50% of cultured adult AMC (16, 20). Additionally, AMC in slices of adult adrenals may exhibit a hypoxia-induced increase in intracellular Ca2+ of similar magnitude to that observed in cells from neonatal slices (30). Interestingly, 10 nM ryanodine, which releases Ca2+ from the endoplasmic reticulum, prolonged the hypoxia-induced Ca2+ rise in adultbut not neonatal cells, suggesting that Ca2+-induced Ca2+ release may contribute to the sustained Ca2+rise is adult cells (30). The presence of 02-sensitive responses in adult AMC is in contrast with our previous report where only 1/27 juvenile cells responded significantly to hypoxia as determined by K+ current inhibition (33). Unfortunately, it was not determined if hypoxia inhibited outward currents or caused membrane depolarization of adult AMC in adrenal slices.

How can the presence of 02-sensing in adult AMC be reconciled with its absence in juvenile cells? It seems reasonable to suggest that in long-term culture experiments, there was a return ofthe non-neurogenic 02-sensing mechanism, similar to the effects of denervating adult AMC in vivo (28). However, this point is more difficult to reconcile in the studies that used cells maintained in short-term culture or in slice preparations. One possible explanation is that there is a quantitative difference in the number of juvenile and adult AMC that respond to hypoxia. Such a difference could arise if the number of functional synapses between splanchnic nerve endings and AMC are culled during maturation, so that there are fewer in the adult than juvenile adrenal gland. Indeed, it has been reported that innervation ofthe adrenal gland develops postnatally (11). One further potential explanation for the observed 02-sensitivity of adult AMC in slices is the potentiation ofthe hypoxic responses due to hyperoxic exposure. It is routine in slice preparations to bubble tissue sections in 95-100% 02 for ~ 1 hr, and exposure to hyperoxia has been reported to augment the hypoxic ventilatory response of rats in a nitric oxide (NO)-dependent manner (10). Thus, it is conceivable that exposure to hyperoxia alters the sensitivity of AMC to hypoxia.

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Pregnancy Diet Plan

Pregnancy Diet Plan

The first trimester is very important for the mother and the baby. For most women it is common to find out about their pregnancy after they have missed their menstrual cycle. Since, not all women note their menstrual cycle and dates of intercourse, it may cause slight confusion about the exact date of conception. That is why most women find out that they are pregnant only after one month of pregnancy.

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