Focal photodynamic intracellular acidification as a cancer therapeutic.

Cancer cells utilize an array of proton transporters to regulate intra- and extracellular pH to thrive in hypoxic conditions, and to increase tumor growth and metastasis. Efforts to target many of the transporters involved in cancer cell pH regulation have yielded promising results, however, many productive attempts to disrupt pH regulation appear to be non-specific to cancer cells, and more effective in some cancer cells than others. Following a review of the status of photodynamic cancer therapy, a novel light-activated process is presented which creates very focal, rapid, and significant decreases in only intracellular pH (pHi), leading to cell death. The light-activation of the H+ carrier, nitrobenzaldehyde, has been effective at initiating pH-induced apoptosis in non-cancerous and numerous cancerous cell lines in vitro, to include breast, prostate, and pancreatic cancers. Also, this intracellular acidification technique caused significant reductions in tumor growth rate and enhanced survival in mice bearing triple negative breast cancer tumors. The efficacy of an NBA-upconverting nanoparticle to kill breast cancer cells in vitro is described, as well as a discussion of the potential intracellular mechanisms underlying the pH-induced apoptosis.

Intracellular acidosis and pH regulation in central respiratory chemoreceptors.

Dysfunctions of brainstem regions responsible for central CO2 chemoreception have been proposed as an underlying pathophysiology of Sudden Infant Death Syndrome (SIDS). We recorded respiratory motor output and intracellular pH (pHi) from chemosensitive neurons in an in vitro tadpole brainstem during normocapnia and hypercapnia. Flash photolysis of the H+ donor nitrobenzaldehyde was used to induce focal decreases in pHi alone. Hypercapnia and flash photolysis significantly decreased pHi from normocapnia. In addition, chemoreceptors did not regulate pHi during hypercapnia, but demonstrated significant pHi recovery when only pHi was reduced by flash photolysis. Respiration was stimulated by decreases in pHi (hypercapnia and flash photolysis) by decreases in burst cycle. These data represent our ability to load the brainstem with nitrobenzaldehyde without disrupting the respiration, to quantify changes in chemoreceptor pHi recovery, and to provide insights regarding mechanisms of human health conditions with racial/ethnic health disparities such as SIDS and Apnea of Prematurity (AOP).

Optical recording of intracellular pH in respiratory chemoreceptors.

We studied the spontaneously active in vitro tadpole brainstem and recorded whole nerve respiratory activity while simultaneously visualizing intracellular pH (pHi) dynamics using the pH-sensitive dye, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF, AM). The isolated, superfused tadpole brainstem is well oxygenated and retains synaptic connectivity among respiratory central pattern generators, central respiratory chemoreceptors, and respiratory motor neurons. We generated a calibration curve to correlate the emitted fluorescence of BCECF to pHi. In addition, we demonstrated that the dye loading protocol that we established labeled an adequate number of cells and did not disrupt spontaneous respiratory rhythmogenesis or the respiratory response to central chemoreceptor stimulation. Validation of the use of the pH sensitive dye BCECF in this preparation will permit further characterization of the pH regulatory responses of central respiratory chemoreceptors and allow correlation between the changes in pHi in central chemoreceptors and respiratory motor output recorded from cranial nerves.

Serotonergic modulation of respiratory rhythmogenesis and central chemoreception.

In addition to evidence supporting serotonergic modulation of respiratory rhythmogenesis, serotonergic mechanisms play a role in central respiratory chemoreception. We examined the role of serotonin 5HT1A receptors in respiratory rhythmicity and central respiratory chemosensitivity in in vitro brainstem preparations of the bullfrog tadpole, Rana catesbeiana. Spontaneous respiratory motor output was recorded from cranial nerve 7 at control bath pH (7.8) and hypercapnic bath pH (7.4) as bath concentrations of a 5HT1A receptor agonist were steadily increased from 0.5 to 25 microM. Activation of the 5HT1A receptor significantly altered the respiratory burst cycle. Significant increases in both gill and lung burst cycle were observed in response to bath application of 8-OH-DPAT; gill burst cycle in response to 8-OH-DPAT was influenced by bath pH, as gill burst cycle at bath pH 7.8 was not significantly increased at 0.5 or 5.0 microM 8-OH-DPAT. However, when the pH was reduced to 7.4 gill burst cycle was significantly increased at these same bath concentrations of 8-OH-DPAT. Gill burst amplitude was not altered in response to bath application of 8-OH-DPAT; however, lung burst amplitude was significantly decreased at 25.0 microM 8-OH-DPAT at bath pH 7.8. These data indicate that 5HT1A receptors are involved in neural respiratory rhythmogenic and chemoreceptive circuits in the bullfrog tadpole, and support the hypothesis that abnormalities in serotonergic systems may be an underlying component of Sudden Infant Death Syndrome.

Effect of prevention of lung inflation on metamorphosis and respiration in the developing bullfrog tadpole, Rana catesbeiana.

We tested the hypothesis that respiratory development would be retarded in tadpoles reared in aquaria in which a barrier prevented access to the air-water interface. To test this hypothesis, we examined swimming behavior and respiration in intact tadpoles and gill and lung respiratory activity and central chemosensory responses in an in vitro brainstem preparation. The "barrier" tadpoles had significantly lower resting gill frequencies and higher lung breath attempts than control tadpoles at the same metamorphic stage. Control tadpoles swam greater distances and spent more time in the upper one third of the aquaria, while barrier tadpoles spent significantly more time at the bottom of the aquaria. There was significantly greater mortality for barrier tadpoles compared to control animals in the earliest and latest metamorphic stages. Mean body weight was significantly greater, and metamorphic rate was reduced in barrier tadpoles. Neither control nor barrier tadpole brainstem preparations demonstrated a gill ventilatory response to CO(2); however, both control and barrier preparations possessed significant lung frequency responses to central CO(2) chemoreceptor stimulation. Bath application of the GABA(A) and glycine receptor antagonists, bicuculline and strychnine, had greater effects on control tadpole gill burst activity and produced a similar large-amplitude bursting pattern in both control and barrier tadpoles, that was insensitive to CO(2) chemoreceptor stimulation. We conclude that development of the respiratory pattern was perturbed by the barrier, but the major effect was on gill ventilation rather than lung ventilation as we had expected.

Ontogeny of central CO2 chemoreception: chemosensitivity in the ventral medulla of developing bullfrogs.

Sites of central CO2 chemosensitivity were investigated in isolated brain stems from Rana catesbeiana tadpoles and frogs. Respiratory neurograms were made from cranial nerve (CN) 7 and spinal nerve 2. Superfusion of the brain stem with hypercapnic artificial cerebrospinal fluid elicited increased fictive lung ventilation. The effect of focal perfusion of hypercapnic artificial cerebrospinal fluid on discrete areas of the ventral medulla was assessed. Sites of chemosensitivity, which are active continuously throughout development, were identified adjacent to CN 5 and CN 10 on the ventral surface of the medulla. In early- and middle-stage tadpoles and frogs, unilateral stimulation within either site was sufficient to elicit the hypercapnic response, but simultaneous stimulation within both sites was required in late-stage tadpoles. The chemosensitive sites were individually disrupted by unilateral application of 1 mg/ml protease, and the sensitivity to bath application or focal perfusion of hypercapnia was reassessed. Protease lesions at CN 10 abolished the entire hypercapnic response, but lesions at CN 5 affected only the hypercapnic response originating from the CN 5 site. Neurons within the chemosensitive sites were also destroyed by unilateral application of 1 mM kainic acid, and the sensitivity to bath or focal application of hypercapnia was reassessed. Kainic acid lesions within either site abolished the hypercapnic response. Using a vital dye, we determined that kainic acid destroyed neurons by only within 100 microm of the ventral medullary surface. Thus, regardless of developmental stage, neurons necessary for CO2 sensitivity are located in the ventral medulla adjacent to CN 5 and 10.

Central CO2 chemoreception in developing bullfrogs: anomalous response to acetazolamide.

Central CO(2) chemoreception and the role of carbonic anhydrase were assessed in brain stems from Rana catesbeiana tadpoles and frogs. Buccal and lung rhythms were recorded from cranial nerve VII and spinal nerve II during normocapnia and hypercapnia before and after treatment with 25 microM acetazolamide. The lung response to acetazolamide mimicked the hypercapnic response in early-stage and midstage metamorphic tadpoles and frogs. In late-stage tadpoles, acetazolamide actually inhibited hypercapnic responses. Acetazolamide and hypercapnia decreased the buccal frequency but had no effect on the buccal duty cycle. Carbonic anhydrase activity was present in the brain stem in every developmental stage. Thus more frequent lung ventilation and concomitantly less frequent buccal ventilation comprised the hypercapnic response, but the response to acetazolamide was not consistent during metamorphosis. Therefore, acetazolamide is not a useful tool for central CO(2) chemoreceptor studies in this species. The reversal of the effect of acetazolamide in late-stage metamorphosis may reflect reorganization of central chemosensory processes during the final transition from aquatic to aerial respiration.