Augmentation of hypoxic respiration after brief hyperoxia in the anesthetized cat: Putative function of GABA<Subscript>A</Subscript> neurotransmission

In this study, we attempted to determine the role of GABA neurotransmission in augmentation of hypoxic respiration by antecedent hyperoxic breathing. The experiments were performed in anesthetized, paralyzed and vagotomized cats divided into control and bicuculline (a GABA_A receptor blocker)-injected groups. The experimental protocol consisted of exposing the animals to successive hypoxic-hyperoxic-hypoxic conditions. Respiration was assessed using phrenic electroneurograms, from which the peak phrenic height, a surrogate of the tidal volume component, and respiratory rate were obtained, and their product, the respiratory minute output, was calculated. We found that prior hyperoxic ventilation increased the subsequent respiratory response to hypoxia by an average of 23.5%, compared with the preoxygen response. This increase was driven by volume respiration. The biphasic character of the hypoxic respiratory response, consisting of stimulatory and depressant phases, was sustained. Bicuculline abolished the augmentative effect on hypoxic respiration of prior hyperoxia, which suggests that oxygenation induces GABA_A-mediated hyperexcitability of respiratory neurons, possibly by the liberation of reactive oxygen species. We concluded that GABA neurotransmission is pertinent to the effect of hyperoxia on hypoxic respiratory reactivity.     

Response of mitochondrial antioxidant system and respiratory pathways to reactive nitrogen species in pea leaves

Nitric oxide (NO) has emerged as an important signaling molecule in plants, but little is known about the effects of reactive nitrogen species in plant mitochondria. In this study, the effects of DETA‐NONOate, a pure NO slow generator, and of SIN‐1 (3‐morpholinosydnonimine), a peroxynitrite producer, on the activities of respiratory pathways, enzymatic and non‐enzymatic antioxidants have been investigated in isolated mitochondria from pea leaves. No significant changes in lipid peroxidation, protein oxidation or in ascorbate and glutathione redox state were observed after DETA‐NONOate treatments whereas cytochrome pathway (CP) respiration was reversibly inhibited and alternative pathway (AP) respiration showed little inhibition. On the other hand, NO did not affect neither activities of Mn superoxide dismutase (Mn‐SOD) nor enzymes involved in the ascorbate and glutathione regeneration in mitochondria except for ascorbate peroxidase (APX), which was reversely inhibited depending on ascorbate concentration. Finally, SIN‐1 treatment of mitochondria produced a decrease in CP respiration, an increase in protein oxidation and strongly inhibited APX activity (90%), with glutathione reductase and dehydroascorbate reductase (DHAR) being moderately inhibited (30 and 20%, respectively). This treatment did not affect monodehydroascorbate reductase (MDHAR) and Mn‐SOD activities. Results showed that mitochondrial nitrosative stress was not necessarily accompanied by oxidative stress. We suggest that NO‐resistant AP and mitochondrial APX may be important components of the H₂O₂‐signaling pathways under nitrosative stress induced by NO in this organelle. Also, MDHAR and DHAR, via ascorbate regeneration, could constitute an essential antioxidant defense together with Mn‐SOD, against NO and ONOO^? stress in plant mitochondria.     

Differences in time-dependent hypoxic phrenic responses among inbred rat strains.

Hypoxic ventilatory responses differ between rodent strains, suggesting a genetic contribution to interindividual variability. However, hypoxic ventilatory responses consist of multiple time-dependent mechanisms that can be observed in different respiratory motor outputs. We hypothesized that strain differences would exist in discrete time-dependent mechanisms of the hypoxic response and, furthermore, that there may be differences between hypoglossal and phrenic nerve responses to hypoxia. Hypoglossal and phrenic nerve responses were assessed during and after a 5-min hypoxic episode in anesthetized, vagotomized, and ventilated rats from four inbred strains: Brown Norway (BN), Fischer 344 (FS), Lewis (LW), and Piebald-viral-Glaxo (PVG). During baseline, burst frequency was higher in PVG than LW rats (P < 0.05), phrenic burst amplitude was higher in PVG vs. other strains (P < 0.05), and hypoglossal burst amplitude was higher in PVG and BN vs. FS and LW (P < 0.05). During hypoxia, burst frequency did not change in BN or LW rats, but it increased in PVG and FS rats. The phrenic amplitude response was smallest in PVG vs. other strains (P < 0.05), and the hypoglossal response was similar among strains. Short-term potentiation posthypoxia was slowest in FS and fastest in LW rats (P < 0.05). Posthypoxia frequency decline was absent in PVG, but it was observed in all other strains. Augmented breaths were observed during hypoxia in FS rats only. Thus genetic differences exist in the time domains of the hypoxic response, and these are differentially expressed in hypoglossal and phrenic nerves. Furthermore, genetic diversity observed in hypoxic ventilatory responses in unanesthetized rats may arise from multiple neural mechanisms.     

Chronic hypoxia abolishes posthypoxia frequency decline in the anesthetized rat

In anesthetized rats, increases in phrenic nerve amplitude and frequency during brief periods of hypoxia are followed by a reduction in phrenic nerve burst frequency [posthypoxia frequency decline (PHFD)]. We investigated the effects of chronic exposure to hypoxia on PHFD and on peripheral and central O2-sensing mechanisms. In Inactin-anesthetized (100 mg/kg) Sprague-Dawley rats, phrenic nerve discharge and arterial pressure responses to 10 s N2 inhalation were recorded after exposure to hypoxia (10 +/- 0.5% O2) for 6-14 days. Compared with rats maintained at normoxia, PHFD was abolished in chronic hypoxic rats. Because of inhibition of PHFD, the increased phrenic burst frequency and amplitude after N2 inhalation persisted for 1.8-2.8 times longer in chronic hypoxic (70 s) compared with normoxic (25-40 s) rats (P < 0.05). After acute bilateral carotid body denervation, N2 inhalation produced a short depression of phrenic nerve discharge in both chronic hypoxic and normoxic rats. However, the degree and duration of depression of phrenic nerve discharge was smaller in chronic hypoxic compared with normoxic rats (P < 0.05). We conclude that after exposure to chronic hypoxia, a reduction in PHFD contributes to an increased duration of the acute hypoxic ventilatory response in anesthetized rats. Furthermore, after exposure to chronic hypoxia, the central network responsible for respiration is more resistant to the depressant effects of acute hypoxia in anesthetized rats.     

Effect of Short-Term Intermittent Normobaric Hypoxia on the Regulation of External Respiration in Humans

Baseline external respiration and gas exchange values, as well as ventilatory thresholds and sensitivity to the O₂ and CO₂ stimuli in hypoxic and hypercapnic tests, were measured 1 h before and after a session of intermittent normobaric hypoxia (INH) (six repetitions with a 5-min inhalation of a gas mixture (10% O₂) alternating with a 3-min inhalation of atmospheric air). After an INH session, the background CO₂ level in the lungs increased by 10%. In the hypercapnic test, the actuation threshold of the ventilatory response did not change, whereas ventilatory sensitivity increased. The maximal pulmonary ventilation and the corresponding critical CO₂ level in the lungs also increased at the end of the test. In the hypoxic test, the ventilatory response occurred at a decreased level of blood oxygenation after an INH session, the pulmonary ventilation level being decreased and the CO₂ content in the lungs being increased at the end of the test. The data obtained evidence the maintenance of changed gas homeostasis for 1 h after an INH session. In this process, control of respiration was effected, with the hypoxic drive being weakened and the peripheral chemoreceptor sensitivity being decreased. The hypercapnic drive also increased, which may be determined by readjustment in the central mechanisms of respiratory regulation.     

Absence of MK801-induced inspiratory prolongation in chronically hypoxic rats.

N-methyl-D-aspartate (NMDA) receptors play important roles in the neural control of respiration. We hypothesized that the brainstem circuit for respiratory control is modulated in response to chronic hypoxia during postnatal maturation, and the modulation may involve changes in the neurotransmission mediated by the NMDA receptors for inspiratory termination. Electrophysiological studies were performed on anesthetized, vagotomized, paralyzed and ventilated rats. Phrenic nerve activity was recorded in normoxic control and chronically hypoxic (CH) rats maintained in normobaric hypoxia (10% O2) for 4-5 weeks from birth. In normoxic rats, the NMDA receptor antagonist, dizocilpine (MK801, i.p.) irreversibly increased inspiratory time (Ti) by 53% and decreased expiratory time (Te) by 29%. However, MK801 did not change the Ti, Te, respiratory rate and peak phrenic nerve activity in CH rats. Results suggest that brainstem mechanisms underlying inspiratory termination mediated by NMDA receptors are modulated by early chronic hypoxia.     

Ventilatory Chemosensory Drive Is Blunted in the mdx Mouse Model of Duchenne Muscular Dystrophy (DMD)

Duchenne Muscular Dystrophy (DMD) is caused by mutations in the DMD gene resulting in an absence of dystrophin in neurons and muscle. Respiratory failure is the most common cause of mortality and previous studies have largely concentrated on diaphragmatic muscle necrosis and respiratory failure component. Here, we investigated the integrity of respiratory control mechanisms in the mdx mouse model of DMD. Whole body plethysmograph in parallel with phrenic nerve activity recordings revealed a lower respiratory rate and minute ventilation during normoxia and a blunting of the hypoxic ventilatory reflex in response to mild levels of hypoxia together with a poor performance on a hypoxic stress test in mdx mice. Arterial blood gas analysis revealed low PaO₂ and pH and high PaCO₂ in mdx mice. To investigate chemosensory respiratory drive, we analyzed the carotid body by molecular and functional means. Dystrophin mRNA and protein was expressed in normal mice carotid bodies however, they are absent in mdx mice. Functional analysis revealed abnormalities in Dejours test and the early component of the hypercapnic ventilatory reflex in mdx mice. Together, these results demonstrate a malfunction in the peripheral chemosensory drive that would be predicted to contribute to the respiratory failure in mdx mice. These data suggest that investigating and monitoring peripheral chemosensory drive function may be useful for improving the management of DMD patients with respiratory failure.     

Effect of cadmium on growth and respiration of barley plants grown under two temperature regimes

We studied cadmium effect on the respiratory pathways ratio in the organs of barley (Hordeum distichum L., cv. Novichok) plants grown in water culture at two temperature regimes. Mineral nutrients were supplied daily in exponentially increasing amounts in order to provide for steady-state growth. CdSO₄ (30, 60, or 100 μmol/l) was added to nutrient solution at a single time in the beginning of the exponential growth period (19 days after germination). In further 6 days, the relative growth rate and biomass accumulation declined stronger with the increase in the cadmium concentration in plants grown at 13/8°C (day/night) than at 21/17°C (day/night). Cadmium suppressed root respiration (down to 60% of control) stronger than leaf respiration, and the roots manifested a higher sensitivity to the inhibitor of alternative oxidase, salicylhydroxamic acid. The respiratory pathways ratio in the roots occurred against the background of activated lipid peroxidation (POL). The highest POL activity and the highest proportion of alternative respiration pathway (AP) (up to 46% of total respiration) were observed in the roots in the presence of the highest cadmium concentration (100 μM) under lower temperature (13/8°C). Thus, high cadmium concentrations affected strongly the total rate of respiration and respiratory pathways ratio. Growth temperature modulated Cd effects on respiration. AP activation is one of the mechanisms for maintenance of root cell homeostasis under cadmium-induced stress.     

Ascorbyl palmitate as a carrier of ascorbate into neural tissues

We have investigated the hypothesis that a lipid-soluble derivative of ascorbic acid, ascorbyl-6-palmitate (AP), could serve as a carrier of ascorbate into neural tissues. Ascorbate could then exert its physiological effects in the biomembranes that are the target sites of the cellular signaling pathways which are normally hardly accessible to this water-soluble compound. The potential role of AP would require that it penetrates into tissues. The major objective of the study was to determine whether ascorbate could be recovered from cerebral cortex and carotid body tissues, both sensitive to the hypoxic stimulus, after AP given by gavage. Biological samples were analyzed by HPLC for the determination of ascorbate. We found that ascorbate was recovered from the tissues studied. Its content was higher in both tissues, by nearly an order of magnitude, after ingestion of AP than after ingestion of ascorbic acid, and the ascorbate level was higher in the carotid body than in the cortex. Hypoxia decreased the ascorbate content which implies physiological activity of ascorbate carried alongside the AP molecule. The lipophilic AP was able to cross biological barriers and satisfied the tissue demand for ascorbate better than the hydrophilic form. AP should be considered as the preferred form of transport of ascorbate into neural tissues. The results of this study suggest wider pharmacological applications of ascorbyl palmitate.     

Activities of respiratory pathways and the pool of nonstructural carbohydrates in greening leaves of spring wheat seedlings

Seedlings of spring wheat (Triticum aestivum L.) were used to study the dynamics of leaf respiration, the respiratory pathway ratio, and relation of activities of these pathways to the content of soluble carbohydrates in the leaf during greening of seedlings for 48 h under continuous photosynthetically active light (190 μmol/(m² s)). Changes in leaf respiration during de-etiolation were closely related to modulation of the alternative respiratory pathway (AP) activity. The rate of cytochrome respiratory pathway (CP) depended directly on the carbohydrate content and growth rate. These relations suggest that the substrate regulation of CP activity during greening is mediated by the energy needs for growth and is effectively regulated by the mechanism of respiratory control. The highest rates of AP were observed after a 6-h exposure of seedlings to light. The proportion of CP/AP at this stage was close to unity. The temporal pattern of AP activity during de-etiolation was independent on the content of soluble carbohydrates. Hence, in addition to substrate regulation of AP, there are other intricate mechanisms of AP involvement. Our results are in accordance to the state that the alternative respiratory pathway participates in maintaining homeostasis in phototrophic cells during development of the photosynthetic function.     

Hypoxia-mediated in vivo release of dopamine in nucleus tractus solitarii of rabbits

A wide variety of neuroactive substances have been suggested to be involved in the respiratory depression observed in response to severe hypoxia. By use of the technique of microdialysis, the release of dopamine (DA) was measured in the nucleus tractus solitarii during severe hypoxic provocations (6% O2 in N2) in the adult pentobarbital-anesthetized rabbit. DA release was analyzed by high-performance liquid chromatography with electrochemical detection. Such hypoxic provocations caused pronounced phase of depression in the phrenic nerve activity and enhanced release of DA. After bilateral carotid sinus nerve denervation, acute severe hypoxia did not give rise to enhanced release of DA or to phrenic nerve depression. Mild hypoxic (9% or 12% O2 in N2) or hypercapnic (6% CO2) stimuli resulted in an increased phrenic nerve activity without any concomitant changes in DA release. Decerebration at the midcollicular level in rabbits prevented an enhanced release of DA in the nucleus tractus solitarii during severe hypoxia. The results suggest that 1) DA is involved in the central ventilatory response to severe hypoxia, 2) not only the initial excitatory but also the second depressive phase in response to severe hypoxia is mediated partially by the peripheral chemoreceptors, and 3) the depressive phase is dependent on intact connections from suprapontine structures.     

Brain stem extracellular fluid pH and respiratory drive during hypoxia in newborn pigs

In response to moderate hypoxia many newborn animals are capable of increasing ventilation only transiently. To examine the hypothesis that changes in brain stem extracellular fluid (ECF) pH explain this transient ventilatory response, we measured brain stem ECF pH and respiratory drive during hypoxia in newborn pigs. The animals were anesthetized with alpha-chloralose-urethan, paralyzed, vagotomized, and mechanically ventilated with a servo-controlled ventilator to regulate end-tidal CO2. Hypoxic ventilation for 6 min was achieved by changing inspired gas from 100% to 10-15% O2. Respiration, measured as integrated phrenic nerve activity, showed a range of responses. In 13 trials increased phrenic activity early in the hypoxic period was sustained or further augmented for the duration of the period. In contrast, in eight other trials phrenic activity increased and then declined. Regardless of the respiratory response, ECF pH (measured with a flat-surface electrode) increased slightly (0.009 +/- 0.002 U) during the first 2.5 min of hypoxia and then declined 0.061 +/- 0.017 U by the 6th min. This acidotic shift in ECF pH is inconsistent with the hypothesis that an alkalotic shift causes the nonsustained respiratory response of newborn pigs.     

Calcium as a counteractive agent to streptomycin induced respiratory depression: an in vivo electrophysiological observation.

Effect of streptomycin on respiratory function in cats was studied. It was observed that streptomycin at a dose of 40 mg/kg body weight intravenously (i.v.) caused respiratory failure or streptomycin induced respiratory depression (SIRD). This respiratory failure is not linked with Herring-Breuer stretch receptors because the effect remained unaltered in artificially ventilated cats. The involvement of central structures in SIRD can be discarded since intracarotid and intraventricular administration of streptomycin failed to produce any change in respiration. Studies on monosynaptic reflex, dorsal and ventral root activities of spinal phrenic and intercostal nerves, and on fusimotor and alpha-motor neuron activities of spinal intercostal and phrenic nerves in decerebrated cats indicated clearly that respiratory depression is not only due to blockade at neuromuscular junction but due to functional depression at the level of muscle receptors and spinal cord motor neurons. The respiratory depression induced by streptomycin was more or less completely reversed when calcium was administered intravenously from external source. It is speculated that streptomycin induced respiratory depression may be mediated through calcium inhibition which can be treated with external calcium in conjunction with artificial respiration.     

Mitochondrial respiration at low levels of oxygen and cytochrome c

In the intracellular microenvironment of active muscle tissue, high rates of respiration are maintained at near-limiting oxygen concentrations. The respiration of isolated heart mitochondria is a hyperbolic function of oxygen concentration and half-maximal rates were obtained at 0.4 and 0.7 microM O(2) with substrates for the respiratory chain (succinate) and cytochrome c oxidase [N,N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD)+ascorbate] respectively at 30 degrees C and with maximum ADP stimulation (State 3). The respiratory response of cytochrome c-depleted mitoplasts to external cytochrome c was biphasic with TMPD, but showed a monophasic hyperbolic function with succinate. Half-maximal stimulation of respiration was obtained at 0.4 microM cytochrome c, which was nearly identical to the high-affinity K(')(m) for cytochrome c of cytochrome c oxidase supplied with TMPD. The capacity of cytochrome c oxidase in the presence of TMPD was 2-fold higher than the capacity of the respiratory chain with succinate, measured at environmental normoxic levels. This apparent excess capacity, however, is significantly decreased under physiological intracellular oxygen conditions and declines steeply under hypoxic conditions. Similarly, the excess capacity of cytochrome c oxidase declines with progressive cytochrome c depletion. The flux control coefficient of cytochrome c oxidase, therefore, increases as a function of substrate limitation of oxygen and cytochrome c, which suggests a direct functional role for the apparent excess capacity of cytochrome c oxidase in hypoxia and under conditions of intracellular accumulation of cytochrome c after its release from mitochondria.     

Changes of synaptosomal energy metabolism induced by hypoxia during aging

Synaptosomes were isolated from the motor area of the cerebral cortex of normoxic or hypoxic (PaO₂=17–19 mmHg, for 15 min) beagle dogs of different ages. Synaptosomes were incubated in Krebs-Henseleit-Hepes buffer (for 10 min at 24°C) and the energetic state was defined by: the balance of the labile phosphates (ATP, ADP, AMP, and creatine phosphate); the respiratory rate; the redox state of the intramitochondrial NAD-couple. By the present experimental model, it is possible to evaluate the potential damage (induced by the in vivo hypoxic insult) that synaptosomes cannot reverse under optimal incubation. Aging affected the phosphorylation state of the post-hypoxic incubated synaptosomes. The oxygen consumption rate was quite similar in the synaptosomal fractions from the motor area of hypoxic beagle dogs of different ages, but the cytochromec anda contents were lower in the preparations from hypoxic older brains. In dogs of different ages, hypoxia always lowered the respiration of the synaptosomes, but aging affected the oxygen consumption rates only in post-hypoxic synaptosomes incubated with succinate. The synaptosomal energetic state was defined also by the redox state of the intramitochondrial NAD-couple (G_ox-red) and the phosphorylation state of adenine nucleotide system (G_ATP). The free-energy change (G) for the coupled reactions was calculated, too. In synaptosomes isolated from the cerebral cortex of dogs submitted to hypoxia, the equilibrium (calculated for the mitochondrial electron transfer chain and the phosphorylation of adenine nucleotides) was markedly altered as function of aging. The extensive age-related G changes were largely supported by alteration of the phosphorylation state of adenine nucleotides, rather than by modification of the redox state of the electron transfer chain.All present data suggest that the bioenergetic derangement caused by hypoxia and aging may be interpreted also in terms of modification of the biophysical and biochemical mechanisms involving the mitochondrial membranes and particularly the inner mitochondrial membrane.Special Issue Dedicated to Dr. Abel Lajtha.     

Respiration of wheat root cells under simultaneous inhibition of parts I and III of the electron transport chain of mitochondria by rotenone and antimycine A

Respiration of excised roots of 5 day old wheat seedlings with blocked mitochondrial oxidation under simultaneous action of rotenone and antimycine A was studied. A reduced rate of oxygen uptake was observed within the first hour of root treatment inhibitors. However, after a 5 h exposure there was an increase in oxygen uptake, which was prevented by KCN but amplified by malate and ascorbate. The application of inhibitors caused a considerable increase in the respiratory coefficient (RC) up to 2.1, that suggests a significant CO2 release, when the initial sites of mitochondrial electron transport chain were inhibited. RC did not raise, when ascorbate was added in the presence of inhibitors. We assume that inhibition of mitochondrial oxidation at I and III sites of electron transport chain facilitates switching on the alternative paths of reductant translocation to oxygen. Participation of ATPases and redox system of plasma membrane in the response reactions of respiration directed to the restoration of ion, particularly, proton homeostasis in conditions of inhibited mitochondrial oxidation is discussed.     

Effects of phrenicotomy and exercise on hypoxia-induced changes in phrenic motor output.

To investigate models of plasticity in respiratory motor output, we determined the effects of chronic unilateral phrenicotomy and/or exercise on time-dependent responses to episodic hypoxia in the contralateral phrenic nerve. Anesthetized (urethane), ventilated, and vagotomized rats were presented with three, 5-min episodes of isocapnic hypoxia (11% O(2)), separated by 5 min of hyperoxia (50% O(2)). Integrated phrenic (and hypoglossal) nerve discharge were recorded before and during each hypoxic episode, for the first 5 min after the first hypoxic episode, and at 30 and 60 min after the final episode. Of 36 rats, one-half were sedentary while the other one-half had free access to a running wheel; each of these groups was split into three subgroups: 1) unoperated, 2) chronic left phrenicotomy (27-37 days), and 3) sham operated. Neither unilateral phrenicotomy nor running wheel activity influenced the short-term hypoxic phrenic response (during hypoxia) or long-term facilitation (posthypoxia). Posthypoxia frequency decline was exaggerated in phrenicotomized-sedentary rats relative to unoperated-sedentary rats (change in burst frequency = -23+/-4 vs. -11 +/-5 bursts/min, respectively; 5 min posthypoxia; P<0.05), an effect that was eliminated by spontaneous exercise. The results indicate that neither voluntary running nor unilateral phrenicotomy has major effects on time-dependent hypoxic phrenic responses, with the exception of an unexpected effect of phrenicotomy on posthypoxia frequency decline in sedentary rats.     

Modulation of respiration during brain hypoxia

This review is a summary of the effects of brain hypoxia on respiration with a particular emphasis on those studies relevant to understanding the cellular basis of these effects. Special attention is given to mechanisms that may be responsible for the respiratory depression that appears to be the primary sequela of brain hypoxia in animal models. Although a variety of potential mechanisms for hypoxic respiratory depression are considered, emphasis is placed on changes in the neuromodulator constituency of the respiratory neuron microenvironment during hypoxia as the primary cause of this phenomenon. Hypoxia is accompanied by a net increase in neuronal inhibition due to both decreased excitatory and increased inhibitory neuromodulator levels. A survey of hypoxia-tolerant cellular systems and organisms suggests that hypoxic respiratory depression may be a manifestation of the depression of cellular metabolism, which appears to be a major adaptation to limited oxygen availability in these systems.     

Alternative and cytochrome pathway respiration during shoot bud formation in cultured Pinus radiata cotyledons

Respiration rates for excised cotyledons of Pinus radiata cultured in the presence (shoot-forming) and absence (non-shoot-forming) of N⁶-benzyladenine (BA) over a 21-day period were measured using a Clark-type oxygen electrode. The capacities and activities of cytochrome and alternative pathways of respiration were determined from titrations with KCN (1-10 m M ) and salicylhydroxamic acid (2–20 m M ) individually and in combination. Respiration accounted for by alternative (AP) and cytochrome (CP) pathways varied with both culture treatment and age in culture. Rates of total respiration, CP respiration and AP activity rose concurrent with key developmental events of shoot bud formation. The greatest AP capacity was measured at day 3 in shoot-forming tissue. In contrast, for cotyledons cultured under non-shoot-forming conditions, no AP activity was observed after day 3 despite relatively constant AP capacity throughout the culture period. Although initial increases in cotyledon respiration during the culture period may be related to wounding and introduction to a tissue culture environment, later differences in respiratory patterns between shoot-forming and non-shoot-forming cotyledons appear to be associated with the cytokinin-induced developmental changes which give rise to shoot primordia in cultured radiata pine cotyledons.