Mapping the cellular mechanisms regulating atrial natriuretic peptide secretion

Panu Taskinen

Department of Pharmacology and Toxicology

Abstract

AbstractAtrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) are cardiac hormones, which are involved in the regulation of blood pressure and fluid homeostasis. The major determinant for ANP and BNP release are atrial and ventricular wall stretch, but also some vasoactive factors such as endothelin-1 (ET-1) can enhance cardiac hormone secretion. The mechanical stretch rapidly activates multiple signal transduction pathways in cardiac cells, but the cellular mechanisms mediating stretch-induced ANP secretion are still unknown. The aim of the present study was to examine the cellular mechanisms of autocrine/paracrine factors and stretch-induced ANP secretion.

Genistein, a potent protein tyrosine kinase (PTK) inhibitor, rapidly increased cardiac contractile force and ANP secretion in perfused rat heart. This effect of genistein may be unrelated to the inhibition of PTKs since this stimulation was blocked by a L-type calcium channel antagonist and Ca2+/calmodulin-dependent protein kinase II inhibitor. Pregnancy hormone relaxin increased heart rate and ANP secretion in perfused spontaneously beating heart, suggesting that relaxin may have a role in modulating cardiac function. Cellular mechanisms of atrial wall stretch-induced ANP secretion were also studied. This enhanced secretion was blocked by sarcoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin and PTK inhibitor lavendustin A, indicating that thapsigargin sensitive Ca2+ pools and activation of PTK or PTK cascade have an important role in the regulation of stretch-secretion coupling. In addition, protein phosphatase inhibitor okadaic acid accelerated stretch-induced ANP secretion, suggesting that precise balance of protein kinase and phosphatase activity plays a role in mechanical stretch-induced ANP secretion. Finally interactions of endothelial factors regulating ANP exocytosis were studied. The potent nitric oxide synthase inhibitor L-NAME increased basal and atrial wall stretch-induced ANP secretion in the presence of ET-1, suggesting that nitric oxide may tonically inhibit ANP secretion.

Table of Contents
Acknowledgements
Abbreviations
List of original papers
1. Introduction
2. Review of literature
2.1. Natriuretic peptides and receptors
2.1.1. Natriuretic peptide family
2.1.2. Natriuretic peptide receptors
2.1.3. Physiological effects of natriuretic peptides
2.2. Regulation of ANP secretion
2.2.1. Mechanical factors
2.2.2. Neurohumoral factors and pressor hormones
2.2.3. The endothelial factors, endothelin-1 and nitric oxide
2.3. Signal transduction pathways in cardiac cells
2.3.1. Receptors and G-proteins
2.3.2. Protein kinases
2.3.3. Ionized calcium as a second messenger
2.4. The cellular and molecular response of cardiac myocytes to mechanical stress
2.4.1. Signal transduction pathways activated by mechanical stretch
3. Aims of the research
4. Materials and methods
4.1. Materials
4.2. Animals
4.3. Isolated perfused heart preparation
4.4. Experimental protocols
4.5. Radioimmunoassay of ANP and BNP
4.6. Statistical analysis
5. Results
5.1. Effects of genistein on cardiac function and ANP secretion (I)
5.1.1. Spontaneously beating hearts
5.1.2. Paced hearts
5.1.3. Cellular mechanisms of genistein-induced cardiac effects
5.2. Effects of relaxin on ANP secretion and cardiac function (II)
5.2.1. Cardiac function and ANP secretion
5.2.2. Cellular mechanisms of relaxin-induced cardiac effects
5.3. Effect of thapsigargin on ANP secretion (III)
5.3.1. Basal ANP secretion
5.3.2. Stretch-induced ANP secretion
5.4. Effects of protein kinase inhibitors on stretch-induced ANP and BNP secretion (IV)
5.4.1. Effect of lavendustin A
5.4.2. Effect of okadaic acid
5.4.3. Effects of other protein kinase inhibitors
5.4.4. Sustained increase in atrial wall stretch
5.5. Interaction of nitric oxide and endothelin in ANP secretion (V)
5.5.1. Basal ANP secretion
5.5.2. Atrial stretch-induced ANP secretion
6. Discussion
6.1. Effects of genistein on cardiac function
6.2. Effect of relaxin on ANP secretion
6.3. Mechanical stretch-induced ANP secretion
6.3.1. Role of thapsigargin-sensitive intracellular calcium pools
6.3.2. Role of protein kinases
6.3.3. Role of protein phosphatases
6.3.4. Role of the endothelial factors
7. Summary and conclusions
References
List of Tables
1. Comparison of some properties of natriuretic peptides.
2. Second messenger-dependent protein kinases.
3. Drugs used in the present studies.
List of Figures
1. Some factors that have been shown to affect plasma ANP levels in vivo or stimulate ANP secretion in vitro.
2. Schematic representation of perfused rat heart model.
3. Experimental protocols.
5-1. Effects of relaxin (Rlx) on heart rate and IR-ANP secretion in spontaneously beating isolated perfused rat heart. After a 10-min control period, as shown by the arrows, vehicle or relaxin were added to the perfusion fluid. Values are expressed as mean ± SEM.
5-2. A: Effect of thapsigargin on stretch-induced release of IR-ANP in the isolated, perfused, paced rat hearts. At 10 min, vehicle or thapsigargin were added to the perfusion fluid. The right atrium was distended for 10 min (horizontal line) by elevating the pulmonary artery cannula tip. B: Effect of thapsigargin on the relation between the change of right atrial pressure (RAP) and changes in IR-ANP secretion. ANPstretch indicates ANP secretion during the last 5 minutes of stretching, ANPcontrol before stretching. Values are expressed as mean±SEM. **p<0.01 (Student’s t-test, unpaired).
6. A: Effect of lavendustin A on stretch-induced release of IR-ANP in the isolated, perfused, paced rat hearts. At 10 min, vehicle or lavendustin A were added to the perfusion fluid. The right atrium was distended for 10 min (horizontal line) by elevating the pulmonary artery cannula tip. B: Effect of lavendustin A on the relation between the change of right atrial pressure (RAP) and changes in IR-ANP secretion. ANPstretch indicates ANP secretion during the last 5 minutes of stretching, ANPcontrol before stretching. Values are expressed as mean±SEM. *p<0.05 (Student’s t-test, unpaired).
7. A: Effect of okadaic acid on stretch-induced release of IR-ANP in the isolated, perfused, paced rat hearts. At 10 min, vehicle or okadaic acid were added to the perfusion fluid. The right atrium was distended for 10 min (horizontal line) by elevating the pulmonary artery cannula tip. B: Effect of okadaic acid on the relation between the change of right atrial pressure (RAP) and changes in IR-ANP secretion. ANPstretch indicates ANP secretion during the first 3 minutes of stretching, ANPcontrol before stretching. Values are expressed as mean±SEM. *p<0.05 (Student’s t-test, unpaired).
5-5. Effects of L-NAME on IR-ANP and IR-BNP secretion in the presence of ET-1 in the isolated perfused paced rat hearts. At 10 min, as indicated by the arrows, vehicle (O), ET-1 0.5 nmol/L (), ET-1 0.5 nmol/L plus L-NAME 100 mol/L (o) or ET-1 0.5 nmol/L plus L-NAME 300 mol/L (█) were added to the perfusion fluid. Values are expressed as mean ± SEM.
9. A and B: Effect of L-NAME on atrial wall stretch-induced secretion of IR-ANP in the presence of ET-1 in the isolated perfused paced rat hearts. After a 10 min control period ET-1 (O) at the concentration of 0.2 (A) or 0.5 nmol/L (B) or ET-1 with L-NAME 300 mol/L () was added to the perfusate. The right atrium was distended for 10 min (horizontal line) by elevating the pulmonary artery cannula tip. C and D: Effect of L-NAME on the relation between the change of right atrial pressure (RAP) and changes in IR-ANP secretion. ANPstretch indicates ANP secretion during the first 5 minutes of stretching, ANPcontrol before stretching. Values are expressed as mean±SEM. *p<0.05 (Student"s t-test, unpaired).
10. A hypothetical model of cellular mechanisms of ANP secretion. PKC, protein kinase C; PLC, phospholipase C; DAG, diacylglycerol; G, guanine nucleotide-binding protein.
11. A hypothetical model of interaction of nitric oxide (NO) and endothelin-1 (ET-1) on ANP secretion. NOS, NO synthase; ETX, endothelin receptor subtype; big ET-1, proendothelin-1, cGMP, cyclig guanosine monophosphate.
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